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Ex vivo diagnostics using varied cellular inputs in drug-induced severe cutaneous adverse reactions
  • +11
  • Andrew Awad,
  • Effie Mouhtouris,
  • Catriona Vi Nguyen-Robertson,
  • Natasha Holmes,
  • Kyra Chua,
  • Ana Copaescu,
  • Fiona James,
  • Michelle Goh S,
  • Ar K. Aung,
  • Dale Godfrey,
  • Elizabeth Philips,
  • Andrew Gibson,
  • Catarina Almeida F,
  • Jason Trubiano
Andrew Awad
Austin Health

Corresponding Author:[email protected]

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Effie Mouhtouris
Austin Health
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Catriona Vi Nguyen-Robertson
The Peter Doherty Institute for Infection and Immunity
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Natasha Holmes
Austin Health
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Kyra Chua
Austin Health
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Ana Copaescu
Austin Health
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Fiona James
Austin Health
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Michelle Goh S
Alfred Health
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Ar K. Aung
Alfred Health
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Dale Godfrey
The Peter Doherty Institute for Infection and Immunity
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Elizabeth Philips
Murdoch University
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Andrew Gibson
Murdoch University
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Catarina Almeida F
The Peter Doherty Institute for Infection and Immunity
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Jason Trubiano
Austin Health
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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.