INTRODUCTION
Drug hypersensitivity reactions (DHRs) are currently a burden on
Healthcare Systems since, although not very frequent, 5-10% of all
adverse drug reactions, they have shown a significant increase in
prevalence over last years in adults and children.1,2Moreover, they can be severe, producing longer patients´ stays and
higher rates of hospital associated infections, requiring the
prescription of alternative drugs that may be less effective, more
toxic, and expensive. It is therefore very important to establish a
correct diagnosis of DHRs avoiding false label of allergy and of
non-allergy, being the latter particularly important for severe DHRs, as
anaphylaxis, Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis
(TEN), and drug reaction with eosinophilia and systemic Symptoms
(DRESS).3
DHRs can be classified according to the time of onset of the symptoms
after drug intake in both immediate (IDHRs) and non-immediate reactions
(NIDHRs). NIDHRs appear more than 1 hour after drug administration. They
show heterogeneous clinical manifestations, ranging from mild
maculopapular exanthemas (MPEs), the most frequent (almost 90% of
cases), to life-threatening as SJS-TEN, or DRESS.4,5The diagnostic procedure is complex including a detailed clinical
history,6 followed by skin tests (STs), patch tests
(PTs) and delayed-reading intradermal tests (IDTs), tests that show low
sensitivity.4,7 Therefore, drug provocation test (DPT)
is in many cases needed for confirming diagnosis; however, it is not
allowed in the evaluation of severe reactions.8 Given
the limitations of STs and DPTs, there is a need for developing
validated in vitro tests to correctly identify the responsible
drug in NIDHRs.
NIDHRs are mainly induced by T-cells through the involvement of
different inflammatory mediators and effector cell
subsets.6,9 Although in most cases T-cells with a Th1
pattern are involved,10 other cell subpopulations can
participate, i.e. cytotoxic T-cells producing soluble Fas-ligand,
perforin/granzyme, granulysin, and TNF-α in SJS-TEN, and other bullous
manifestations11 or Th2 CD4+T-cells
in DRESS12. This highlights the importance for
assessing the effector cellular response to increase the sensitivity ofin vitro tests.13,14
Lymphocyte transformation test (LTT), which determines lymphocyte
proliferation upon drug stimulation, has been widely used to evaluate
NIDHRs with a specificity of 93.9%.15-17 However, the
lack of standardization and the low sensitivity (around 56.1%) has
limited its routine diagnostic use, being largely restricted to research
field.17 Different studies have shown that these data
highly depend on the drug involved and the clinical symptoms, with
higher sensitivity (57.9-88.8%) in mild/moderate
reactions18-20 and lower sensitivity (25-75%) for
severe NIDHR as SJS-TEN13,21-24. This creates the need
for improving the sensitivity of the tests, particularly in severe
reactions with very limited diagnostic approaches
available.13,14,25
Several attempts have been performed for improving LTT sensitivity. The
inclusion of the drug metabolites has shown to be important for some
drugs.26 Other studies have demonstrated that the
inclusion of monocyte-derived dendritic cells (moDCs) as antigen
presenting cells (APCs) improves the drug-specific-cellular
proliferation, and therefore LTT sensitivity, when evaluating patients
with NIDHRs to betalactams (BL), heparins, or radio contrast media
(RCM).19,27,28
The cell proliferative response has been classically measured via the
genome incorporation of tritiated thymidine
(3H).19 This method presents the
disadvantage of using radioactive tracers and of not being able to
discriminate the proliferating subpopulation. Nowadays, the use of
flow-cytometry-based methods, determining the decrease on the content of
fluorescent molecules, such as carboxyfluorescein diacetate succinimidyl
ester (CFSE), into proliferating cells, has allowed us assess not only
the proliferative response but also the possibility of identifying
different cell subtypes, including the effector cells involved in the
reaction.13
The aim of this study was to assess the added value of using
drug-primed-moDCs as APCs and the determination of the proliferative
response of different lymphocyte subpopulations in each clinical
manifestation of NIDHRs using the new LTT approach based on
flow-cytometry technology. To this end, patients with confirmed NIDHR
were evaluated by both conventional and with drug-primed-moDCs LTT
analysing the proliferative response in T-cells and other effector
cells.