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.