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.