1. INTRODUCTION
Respiratory allergies are the most common allergies worldwide, still
increasing in frequency and severity. The World Health Organization
estimated patients suffering from allergic asthma at 235 million in 2013
and the numbers for allergic rhinitis at about 400 million in 2006
[1,2]. Although the number of patients with respiratory allergy
drastically increased on a global scale, the increase is greatest in
Western countries. In 2011, the European Federation of Allergy (EFA)
reported that respiratory allergies affected around 20-30% of the
European population. More specifically, around 68 million patients
suffered from allergic asthma and 113 million from allergic rhinitis
[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
[4]. Alarmingly, a vast majority of individuals affected by
allergies are children and young adults [5]. Although not being
associated with high morbidity and mortality, respiratory allergy 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 vitro by 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, thein 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.