Blister fluid as a cellular input for ex vivo diagnostics in drug-induced severe cutaneous adverse reactions improves sensitivity and explores immunopathogenesis

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 standardization and poor sensitivity. Modern approaches to testing include measurement of IFN-γ release from patient PBMCs stimulated with the suspected causative drug.

Objective We sought to improve ex vivo diagnostics for drug-induced SCARs by comparing enzyme-linked immunospot (ELISpot) sensitivities and flow cytometry–based intracellular cytokine staining and determination of the cellular composition of separate samples (PBMCs or blister fluid cells [BFCs]) from the same donor.

Methods ELISpot and flow cytometry analyses of IFN-γ release were performed on donor-matched PBMC and BFC samples from 4 patients with SCARs with distinct drug hypersensitivity.

Results Immune responses to suspected drugs were detected in both the PBMC and BFC samples of 2 donors (donor patient 1 in response to ceftriaxone and case patient 4 in response to oxypurinol), with BFCs eliciting stronger responses. For the other 2 donors, only BFC samples showed a response to meloxicam (case patient 2) or sulfamethoxazole and its 4-nitro metabolite (case patient 3). Consistently, flow cytometry revealed a greater proportion of IFN-γ–secreting cells in the BFCs than in the PBMCs. The BFCs from case patient 3 were also enriched for memory, activation, and/or tissue recruitment markers over the PBMCs.

Conclusion Analysis of BFC samples for drug hypersensitivity diagnostics offers a higher sensitivity for detecting positive responses than does analysis of PBMC samples. This is consistent with recruitment (and enrichment) of cytokine-secreting cells with a memory/activated phenotype into blisters.