INTRODUCTION
Delayed drug-induced hypersensitivities are a group of presumed
conventional T-cell-mediated reactions that range from mild skin
conditions (e.g. maculopapular exanthema) to severe cutaneous adverse
reactions (SCARs), associated with significant morbidity and
mortality[1]. Traditional in vivo skin testing techniques
such as patch testing (PT) or intradermal testing (DT) are limited by an
absence of standardisation, risk of disease-relapse, and ill-defined
drug testing concentrations[2]. These limitations can impact the
sensitivity of such tests, with published studies suggesting sensitivity
ranging from 58-64% for acute generalised exanthematous pustulosis
(AGEP), 32-80% for drug reaction with eosinophilia and systemic
symptoms (DRESS), and 9-24% for Steven-Johnson syndrome (SJS) and toxic
epidermal necrolysis (TEN)[3]. There is also drug-associated
variability in PT, with beta-lactams displaying higher sensitivities
while allopurinol and its active metabolite, oxypurinol, exhibit very
low sensitivities[4]. Evolving approaches include ex-vivoassays, such as the enzyme-linked immunospot (ELISpot), which detects
interferon-γ (IFN) release following drug challenge. Traditionally
ELISpot assays use the patient’s peripheral blood mononuclear cells
(PBMC) stimulated with the candidate drug to measure cytokine output.
The ELISpot method is advantageous as patients are not subjected to
additional risk through drug re-exposure. Our recent data suggests IFN-
γ release ELISpot is an effective diagnostic tool with a 52-68%
sensitivity and 100% specificity in SCAR patients[5, 6].
ELISpot assays detect cytokine (typically IFN-γ) release, which is
presumed to be produced by CD4+ or CD8+ T cells, in patient PBMC or
blister fluid cells (BFC) following ex vivo stimulation with the
candidate drug. Cytokine secretion is measured as the number of
spot-forming units (SFU)/million cytokine secreting cells. Previous case
reports have suggested a diminished PBMC IFN-γ ELIspot response over
time from SCAR onset, highlighting the importance of performing assays
during the acute phase of drug reactions[7]. This diminished
response in peripheral blood may be associated with the lack of a key
cell population known as tissue resident memory CD8+ T cells (TRM),
which reside within the dermal-epidermal and drug-reactive CD8+ T cells
are gradually lost from peripheral blood during the recovery
period[8]. In contrast, CD8+ TRM cells are more likely to be
recruited into BFC in SCAR patients. One study compared cytokine
production between PBMC and BFC, noting that there was a higher
expression of perforin and granzyme B in BFC[9]. This could be due
to localised skin TRM cells mediating the inflammatory response by
recruiting memory CD8+ T cells from circulation and suggests that
ELISpot assays conducted with PBMC from patients in the late stages of
drug reaction could be less sensitive[6, 7, 10]. Here, we sought to
find ways to improve ex vivo assay sensitivity in SCAR
diagnostics by examining differences in ELISpot results between two
different cellular sources: PBMC and BFC. This study aims to provide
knowledge that will inform future SCAR testing strategies.