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-g release from patient PBMCs stimulated with the suspected causative drug. Objective: We sought to improve ex vivo diagnostics for druginduced 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-g 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-g-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 cytokinesecreting cells with a memory/activated phenotype into blisters.

Time to reinvent the wheel for drug causality diagnostic approaches to severe cutaneous adverse reactions

“…3 Further work is required to understand drug metabolites to increase the sensitivity of these assays, as shown with oxypurinol for allopurinol and 4-nitrosulfamethoxazole for TMP-SMX. 4 Finding pathways to further validate in extended cohorts (i.e. international registries), and then implementing ex vivo and in vitro diagnostics is required.…”

“…Both assays have acceptable sensitivity with ELISpots demonstrating a sensitivity of 52% in SCAR with a specificity of 100% and LTT reported sensitivity of 27%–74% and specificity of 85%–100% 3 . Further work is required to understand drug metabolites to increase the sensitivity of these assays, as shown with oxypurinol for allopurinol and 4‐nitrosulfamethoxazole for TMP‐SMX 4 . Finding pathways to further validate in extended cohorts (i.e.…”

“…3 The two major assays are lymphocyte transformation test (LTT) and enzyme-linked immunospot (ELISpot) assays which measure T-cell response to the implicated drug. 2 Historically, these were performed using the patients' peripheral blood mononuclear cells (PBMCs), but new research has shown increased sensitivity using blister fluid cells (BFCs), 4 varied cut-offs and utilising non-cytotoxic drug concentrations. 5,6 Both assays have acceptable sensitivity with ELISpots demonstrating a sensitivity of 52% in SCAR with a specificity of 100% and LTT reported sensitivity of 27%-74% and specificity of 85%-100%.…”

Extracellular-matrix-mimicked 3D nanofiber and hydrogel interpenetrated wound dressing with a dynamic autoimmune-derived healing regulation ability based on wound exudate

“…Notably, Blister fluid is derived from serum through inflammatory/extravasation process and shares similar components as ECM, such as electrolytes, glucose, cytokines, leukocytes, and metalloproteinases. Above all, antibodies and cytokines in blister fluid are actively adjusted by the immune system according to the wound healing stage [1,10]. However, the bioactivity of blister liquid is restricted by oxidation and bacterial contamination, commonly found in open wounds.…”

Sutton's law and severe cutaneous adverse drug reactions: Is the money in the blister?

“…2 In this issue of the Journal of Allergy and Clinical Immunology: Global , Awad et al suggest that in identifying culprit drugs in severe cutaneous adverse drug reactions (SCARs), “the money” may be in blister cells rather than peripheral blood cells. 3 …”