Blister fluid as a cellular input for ex vivo diagnostics in drug-induced severe cutaneous adverse reactions improves sensitivity and explores immunopathogenesis.
Blister fluid as a cellular input for ex vivo diagnostics in drug-induced severe cutaneous adverse reactions improves sensitivity and explores immunopathogenesis.
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DOI：
10.1016/j.jacig.2021.11.001
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发表时间：
2022-02
期刊：
影响因子：
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通讯作者：
Trubiano, Jason A
中科院分区：
文献类型：
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作者：
Awad, Andrew;Mouhtouris, Effie;Nguyen-Robertson, Catriona Vi;Holmes, Natasha;Chua, Kyra Y L;Copaescu, Ana;James, Fiona;Goh, Michelle S;Aung, Ar Kar;Godfrey, Dale I;Philips, Elizabeth J;Gibson, Andrew;Almeida, Catarina F;Trubiano, Jason A
药物诱导的严重皮肤不良反应（SCAR）被认为是与显著发病率和死亡率相关的T细胞介导的超敏反应。传统的体内测试方法，如贴片或皮内测试，受到缺乏标准化和灵敏度差的限制。现代测试方法包括测量用疑似致病药物刺激的患者PBMC的IFN-γ释放。我们试图通过比较酶联免疫斑点（ELISpot）灵敏度和基于流式细胞术的细胞内细胞因子染色以及测定来自同一供体的单独样品（PBMC或水疱液细胞[BFC]）的细胞组成来改善药物诱导的SCAR的离体诊断。对来自4例具有明显药物超敏反应的SCAR患者的供体匹配PBMC和BFC样品进行IFN-γ释放的ELISpot和流式细胞术分析。在2名供体的PBMC和BFC样本中检测到对可疑药物的免疫应答（供体患者1对头孢曲松有应答，病例患者4对羟嘌呤醇有应答），BFC引起更强的应答。对于其他2名供体，仅BFC样本显示对美洛昔康（病例患者2）或磺胺甲恶唑及其4-硝基代谢产物（病例患者3）有反应。流式细胞术显示BFC中IFN-γ分泌细胞的比例高于PBMC。来自病例患者3的BFC也相对于PBMC富集记忆、活化和/或组织募集标志物。用于药物超敏反应诊断的BFC样本分析提供了比PBMC样本分析更高的检测阳性反应的灵敏度。这与将具有记忆/活化表型的精氨酸分泌细胞募集（和富集）到水泡中一致。
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. 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. 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. 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. 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.