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.