Comparison of Rapid Cytokine Immunoassays for Functional Immune Phenotyping.
Comparison of Rapid Cytokine Immunoassays for Functional Immune Phenotyping.
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DOI：
10.3389/fimmu.2022.940030
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发表时间：
2022
影响因子：
7.3
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中科院分区：
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基于细胞的功能性免疫测定可以允许对患有复杂的异质性免疫疾病（例如脓毒症）的患者进行风险分层。考虑到患者反应的异质性和脓毒症免疫发病机制的不确定性，这些检测必须首先在健康人群中进行定义和校准。我们的目的是比较两种免疫测定的内在一致性和实用性，这两种免疫测定可以提供先天性和适应性免疫应答的替代标记物的数据。我们假设，在鉴定离体全血刺激后肿瘤坏死因子（TNF）和干扰素（IFN）γ产生方面，快速周转、基于微流体的免疫测定（ELLA）与双色酶联免疫斑点（ELISpot）测定相当。这是一项前瞻性、观察性队列分析。将来自10名健康的免疫活性志愿者的全血样品用脂多糖、抗CD 3/抗CD 28抗体或佛波醇12-肉豆蔻酸酯13-乙酸酯与离子霉素刺激4小时或18小时，以分别询问先天性和适应性免疫应答。与ELISpot相比，ELLA分析产生了更精确的TNF和IFNγ浓度测量值，以及TNF和IFNγ浓度的4 - 5个log 10动态范围，而ELISpot的动态范围为2个log 10。无监督聚类准确地预测了通过ELLA分析的90%的样品所使用的离体免疫刺激剂，而通过ELISpot分析的样品为72%。我们首次描述了一种快速而精确的检测方法，用于健康志愿者免疫系统先天和适应性臂的功能询问。ELLA微流控平台的优势可能代表了在产生具有临床实用性的即时检测方面向前迈出的一步，用于识别脓毒症患者的紊乱免疫表型。
Cell-based functional immune-assays may allow for risk stratification of patients with complex, heterogeneous immune disorders such as sepsis. Given the heterogeneity of patient responses and the uncertain immune pathogenesis of sepsis, these assays must first be defined and calibrated in the healthy population. Our objective was to compare the internal consistency and practicality of two immune assays that may provide data on surrogate markers of the innate and adaptive immune response. We hypothesized that a rapid turnaround, microfluidic-based immune assay (ELLA) would be comparable to a dual-color, enzyme-linked immunospot (ELISpot) assay in identifying tumor necrosis factor (TNF) and interferon (IFN)γ production following ex vivo whole blood stimulation. This was a prospective, observational cohort analysis. Whole blood samples from ten healthy, immune-competent volunteers were stimulated for either 4 hours or 18 hours with lipopolysaccharide, anti-CD3/anti-CD28 antibodies, or phorbol 12-myristate 13-acetate with ionomycin to interrogate innate and adaptive immune responses, respectively. ELLA analysis produced more precise measurement of TNF and IFNγ concentrations as compared with ELISpot, as well as a four- to five-log10 dynamic range for TNF and IFNγ concentrations, as compared with a two-log10 dynamic range with ELISpot. Unsupervised clustering accurately predicted the ex vivo immune stimulant used for 90% of samples analyzed via ELLA, as compared with 72% of samples analyzed via ELISpot. We describe, for the first time, a rapid and precise assay for functional interrogation of the innate and adaptive arms of the immune system in healthy volunteers. The advantages of the ELLA microfluidic platform may represent a step forward in generating a point-of-care test with clinical utility, for identifying deranged immune phenotypes in septic patients.