Ungaro Ricardo F, Xu Julie, Kucaba Tamara A, Rao Mahil, Bergmann Christian B, Brakenridge Scott C, Efron Philip A, Goodman Michael D, Gould Robert W, Hotchkiss Richard S, Liang Muxuan, Mazer Monty B, McGonagill Patrick W, Moldawer Lyle L, Remy Kenneth E, Turnbull Isaiah R, Caldwell Charles C, Badovinac Vladimir P, Griffith Thomas S
Sepsis and Critical Illness Research Center and Department of Surgery, University of Florida College of Medicine, Gainesville, FL, United States of America.
Department of Urology, University of Minnesota, Minneapolis, MN, United States of America.
J Immunol Methods. 2024 Oct;533:113743. doi: 10.1016/j.jim.2024.113743. Epub 2024 Aug 13.
Sepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ('SPIES') is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. Heparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFNγ and TNFα production as measured by ELISpot. We demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 h at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 h allowing for a substantial decrease in processing time. The data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.
脓毒症仍然是全球主要的死亡原因，目前尚无经过验证的免疫调节疗法。脓毒症患者免疫内型分层研究（'SPIES'）是一项前瞻性、多中心观察性研究，旨在测试酶联免疫斑点技术（ELISpot）作为一种功能生物测定法的效用，该方法专门用于测量刺激后产生细胞因子的细胞，以识别免疫抑制内型、预测脓毒症患者的临床结局，并测试用于临床开发的潜在免疫刺激剂。大多数ELISpot实验方案要求在将外周血单核细胞（PBMC）纳入检测之前进行分离。相比之下，我们开发了一种稀释全血（DWB）ELISpot实验方案，该方案已在多个实验室得到验证。从健康供体和脓毒症患者中采集肝素化全血，并在不同刺激条件下进行检测，以评估血液稀释、刺激剂浓度、血液储存以及刺激时间对通过ELISpot测量的体外干扰素γ（IFNγ）和肿瘤坏死因子α（TNFα）产生的影响。我们证明了全血稀释的动态范围能够对外源刺激产生强大的体外细胞因子反应。此外，可以使用广泛的刺激剂浓度来诱导细胞因子的产生。进一步的改进表明，抗凝全血在室温下可储存长达24小时而不会显著丧失功能。最后，我们表明体外刺激可以短至4小时，从而大幅减少处理时间。这些数据证明了使用ELISpot在各种刺激条件下测量DWB内细胞功能能力的可行性，以便为临床医生提供脓毒症患者免疫失调程度的信息。
