Ex vivo diagnostics using varied cellular inputs in drug-induced severe
cutaneous adverse reactions
Abstract
Background Drug-induced severe cutaneous adverse
reactions (SCARs) are presumed T-cell-mediated hypersensitivities
associated with significant morbidity and mortality. Traditional
in-vivo testing methods, such as patch or intradermal testing,
are limited by a lack of standardisation and poor sensitivity. Modern
approaches to testing include measurement of IFN-γ release from patient
peripheral blood mononuclear cells (PBMC) stimulated with the suspected
causative drug. Objective We sought to improve
ex-vivo diagnostics for drug-induced SCAR by comparing
enzyme-linked immunospot (ELISpot) sensitivities and flow
cytometry-based intracellular cytokine staining (ICS) and cellular
composition of separate samples (PBMC or blister fluid cells (BFC)) from
the same donor. Methods IFN-γ release ELISpot and flow
cytometry analyses were performed on donor-matched PBMC and BFC samples
from four SCAR patients with distinct drug-allergies.
Results Immune responses to suspected drugs were
detected in both PBMC and BFC samples of two donors (Case 1 in response
to ceftriaxone and Case 4 to oxypurinol), with BFC eliciting stronger
responses. For two other donors, only BFC samples showed a response to
meloxicam(Case 2) or sulfamethoxazole and its 4-Nitro metabolite (Case
3). Consistently, flow cytometry revealed a greater proportion of
IFN-γ-secreting cells in the BFC compared to PBMC. BFC cells from Case 3
were also enriched for memory/activation/tissue-recruitment markers over
PBMC. Conclusion Analysis of BFC samples for
drug-allergy diagnostics offers a higher sensitivity for detecting
positive responses compared to PBMC. This is consistent with recruitment
(and enrichment) of cytokine-secreting cells with a memory/activated
phenotype into blisters.