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.