DISCUSSION
The diagnosis of NIDHRs involves a great complexity due to the existence of different clinical manifestations related to the involvement of many pathomechanisms and the existence of severe reactions which difficult the application of clinical procedures. Moreover, in vivo tests such as STs have a doubtful value to evaluate NIHDR, due to their low sensitivity and because for some drugs its use is not recommended or not available. For this reason, DPT is the gold-standard, although it is not risk-free, and for most severe clinical entities it is not allowed.36
The implementation of in vitro tests with good sensitivity in the clinical practice would be a crucial step in the improvement of NIDHR diagnosis, especially for severe cases. Among others, LTT is a widely used tool for assessing specific proliferation of cell populations in response to a concrete drug. Traditionally, this proliferation has been measured by the uptake of 3H-thymidine and was measured by radioactivity, making it impracticable for routine laboratories.37 The mean sensitivity of LTT with PBMCs ranges from 60% to 70%.38 Considering different clinical manifestations, it has been observed to be higher in mild-moderate reactions (65.1%) than in severe ones (39.9%).24
In our study, including patients with different clinical manifestations and drugs involved, we obtained a low sensitivity (33%) in the C-LTT, which agrees with previous studies that also include a wide panel of clinical symptoms.18,21 However, other studies, also with different clinical symptoms, reported a higher sensitivity.23,39,40 The main difference could be that, in these latter works, the responsible drugs are mainly BLs and anticonvulsants, which have shown a higher sensitivity in LTT.13 All of this indicates that conventional LTT with PBMCs does not show optimal sensitivity.
It has been reported that the inclusion of professional APCs could improve the sensitivity of LTT as shown in NIDHRs to BLs, heparins, and RCM.19,27,28 In our study, comparisons between C-LTT with the test using DCs (dDC-LTT) showed an important increase in general sensitivity from 33.3% to 65%.
An important issue is the capacity of in vitro test for evaluating different clinical manifestations, since in previous studies LTT reported higher sensitivity in mild-moderate NIDHR reactions than in severe ones.13,14,20,22 However, in our study we found different results with lower sensitivity in MPE (14.5%) and better sensitivity in severe reactions as SJS-TEN (62.5%). Although we do not know the real reasons, the different drugs involved in the reactions in each study could be a factor for these discrepant results. Moreover, the inclusion of moDCs in the LTT significantly increases the sensitivity to 87.5% in SJS-TEN and 41.6% in MPE, with no changes in specificity (85%). These data strongly show the beneficial effect of including mo-DCs for amplifying the specific immunological response and specially for improving the results when evaluating patients with severe reactions as SJS-TEN or AGEP for which LTT has classically shown a low sensitivity.
On the other hand, different studies have stated that focussing on the effector response will help increase the sensitivity of in vitrocellular tests.13,14 This has been analysed by determining different inflammatory mediators by flow-cytometry and ELISpot, however with heterogeneous results.20,21,23,37,41 Therefore, since no in vitro test produces enough sensitivity, other authors recommend the combination of different assays to evaluate NIDHRs.21,22,37
The use of flow-cytometry technology could represent a novel approach that allows the evaluation in routine laboratories. Preliminary studies have shown the possibility of evaluating DHRs by measuring the upregulation of CD69 by T-cells15 or cytokine production37 after stimulation with the suspected drug. However, little is known about the utility of measuring the proliferation response by using CFSE. One important advantage of measuring the CFSEdim for the proliferative response by flow-cytometry is the direct possibility of measuring the proliferation of different cell subpopulations involved, including those with low rates but important implications.42 In our study, we tried to evaluate the effect response by analysing the differential proliferative response of different cell subpopulations, showing that CD4 T-lymphocytes with a Th1 pattern are strongly involved in NIDHRs and their evaluation increases the sensitivity of thein vitro test compared with the evaluation of general T cell, CD3+cells. This was also observed for the different clinical entities included in this study, SJS-TEN, MPE, and AGEP, indicating the participation of this cell subset in the pathomechanism involved in NIHDRs.9 The other important cell subpopulation was NK cells, which have shown to be involved in all clinical manifestations although with differences regarding the NK subpopulations with higher proliferation of inflammatory NK (NKIFN-γ) in MPE as previously described11, and cytotoxic NK (NKPerf) in SJS-TEN according to the mechanism involved in these reactions43. Interestingly, CD4+T-lymphocytes with a Th2 pattern have shown to be involved mainly in MPE as previously described.9,21 With all these data and including the effector cell subpopulations involved in each reaction, CD3++CD4+Th1 cells in SJS-TEN, CD3++CD4+Th1+NK cells in MPE and CD3++NK cells in AGEP, we could significantly increase the overall sensitivity of the in vitrotest to 87% with 85% of specificity. Importantly, this increase in sensitivity was achieved with the performance of a unique in vitro test.
In summary, these data indicate that in vitro test analysing the proliferative response to drugs in NIDHRs can be highly improved by presenting the drug by professional APC as moDCs and focussing on the subpopulations participating in the immunological mechanism for each clinical manifestation in a specific manner. This can be easily achieved thanks to the use of flow-cytometry-based tests. Further advances on the knowledge of the mechanism and the identification of specific biomarkers that will be included in the test will increase the in vitrodiagnosis of NIDHRs.