Bonavia Anthony S, Samuelsen Abigail, Liang Menglu, Hanson Jodi, McKeone Daniel, Chroneos Zissis C, Halstead E Scott
Department of Anesthesiology and Perioperative Medicine, Penn State Milton S. Hershey Medical Center, Hershey, PA, USA.
Department of Epidemiology and Biostatistics, School of Public Health, University of Maryland, Baltimore, MD, USA.
Intensive Care Med Exp. 2023 Oct 13;11(1):70. doi: 10.1186/s40635-023-00556-w.
Sepsis is characterized by highly heterogeneous immune responses associated with a spectrum of disease severity. Methods that rapidly and sensitively profile these immune responses can potentially personalize immune-adjuvant therapies for sepsis. We hypothesized that the ELLA microfluidic approach to measure cytokine production from the whole blood of septic and critically ill patients would deliver faster, more precise results than the existing optic-driven ELISpot quantification. We tested our hypothesis by measuring ex vivo-stimulated production of TNF and IFNγ in critically ill and septic patients (n = 22), critically ill and non-septic patients (n = 10), and healthy volunteers (n = 10) through both ELLA and ELISpot immunoassays. Blood samples were subjected to one of three stimulants for 4 h or 18 h durations during days 1, 7-10, and 14 of critical illness. Stimulants for lymphocytes included anti-CD3/anti-CD28 and phorbol 12-myristate 13-acetate (PMA), whereas LPS was used for monocytes. Stimulated TNF and IFNγ concentrations were then associated with 30-day mortality.
Both ELISpot and ELLA immunoassays showed substantial agreement in TNF concentrations post 4 h and 18 h LPS stimulation, with concordance correlation coefficients at 0.62 and 0.60, respectively. ELLA had a broad dynamic measurement range and provided accurate TNF and IFNγ readings at both minimal and elevated cytokine concentrations (with mean coefficients of variation between triplicate readings at 2.1 ± 1.4% and 4.9 ± 7.2%, respectively). However, there was no association between the ELLA-determined cytokine concentrations on the first day of critical illness and 30-day mortality rate. In contrast, using the ELISpot for cytokine quantification revealed that non-survivors had reduced baseline TNF levels at 18 h, decreased LPS-induced TNF levels at 18 h, and diminished TNF levels post 4 h/18 h anti-CD3/28 stimulation.
Our study affirms the feasibility of obtaining dependable immune phenotyping data within 6 h of blood collection from critically ill patients, both septic and non-septic, using the ELLA immunoassay. Both ELLA and ELISpot can offer valuable insights into prognosis, therapeutic strategies, and the underlying mechanisms of sepsis development.
脓毒症的特征是免疫反应高度异质性，与一系列疾病严重程度相关。能够快速且灵敏地分析这些免疫反应的方法可能会使脓毒症的免疫辅助治疗实现个体化。我们假设，采用ELLA微流控方法测量脓毒症患者和危重症患者全血中的细胞因子产生情况，会比现有的光学驱动酶联免疫斑点法（ELISpot）定量分析得出结果更快、更精确。我们通过ELLA和ELISpot免疫测定法，检测了危重症和脓毒症患者（n = 22）、危重症和非脓毒症患者（n = 10）以及健康志愿者（n = 10）体外刺激产生的肿瘤坏死因子（TNF）和干扰素γ（IFNγ），以此验证我们的假设。在危重症的第1天、第7 - 10天和第14天，采集的血样分别接受三种刺激物之一刺激4小时或18小时。淋巴细胞刺激物包括抗CD3/抗CD28和佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（PMA），而脂多糖（LPS）用于刺激单核细胞。然后将刺激后的TNF和IFNγ浓度与30天死亡率相关联。
ELISpot和ELLA免疫测定法均显示，在LPS刺激4小时和18小时后，TNF浓度具有高度一致性，一致性相关系数分别为0.62和0.60。ELLA具有广泛的动态测量范围，在细胞因子浓度最低和升高时均能提供准确的TNF和IFNγ读数（三次重复读数的平均变异系数分别为2.1±1.4%和4.9±7.2%）。然而，危重症第1天通过ELLA测定的细胞因子浓度与30天死亡率之间并无关联。相比之下，使用ELISpot进行细胞因子定量分析发现，未存活者在18小时时基线TNF水平降低，18小时时LPS诱导的TNF水平降低，以及在4小时/18小时抗CD3/28刺激后TNF水平降低。
我们的研究证实，使用ELLA免疫测定法，在采集脓毒症和非脓毒症危重症患者血液后6小时内获得可靠的免疫表型数据是可行的。ELLA和ELISpot都能为脓毒症的预后、治疗策略及发病机制提供有价值的见解。
