危重病人全血内毒素血症检测的内毒素活性测定
Summary
我们特此提出一个方案,在床边测量人类全血样本的内毒素活性。内毒素活性测定是一个简单的测试,可能是脓毒症危重病人的一个有用的生物标志物。
Abstract
脂多糖,也称为内毒素,是革兰氏阴性细菌的基本成分,在败血症和败血症休克的发展中起着至关重要的作用。早期发现正在迅速演变为严重疾病的传染性过程可能会促使更快和更密集的治疗,从而可能导致更好的患者结果。内毒素活性(EA)测定可在床边用作系统内毒素血症的可靠生物标志物。内毒素活性水平升高的检测已反复证明与败血症和败血症休克患者疾病严重程度增加有关。测定快速且易于执行。简单地说,取样后,全血的等分与抗内毒素抗体混合,并加入LPS。内毒素活性被测量为通过化学发光检测到的含以结束中性粒细胞的相对氧化爆发。测定的输出以从0(不存在)到1(最大)的刻度表示,并分为”低”(<0.4单位)、"中间"(0.4~0.59个单位)或"高"(±0.6个单位)。本手稿报告了实施 EA 测定的详细方法和理由。
Introduction
脂多糖(LPS),也称为内毒素,是革兰氏阴性(GN)细菌膜结构的关键组成部分。它约占细胞壁的10%,对外膜的完整性和平衡至关重要。此外,它是宿主先天免疫系统1,2的有力激活剂。
体外免疫系统细胞在体外接触LPS会导致多个基因的表达发生变化3。在健康人类志愿者中管理非常少量的LPS会触发急性全身炎症的级联,而败血症和败血症休克则可能以较高的内毒素浓度4、5出现。
败血症是一种危及生命的情况,如果不及时识别,可能导致多器官衰竭和死亡。消毒患者必须及时治疗,积极复苏,适当的抗生素治疗,最佳的源头控制和及时的器官支持策略。脓毒症病因的诊断主要基于临床识别和培养性病原体检测6。然而,微生物培养的结果可能长达48小时,在高达30%的病例7中没有结论。早期识别和干预可能导致更好的患者结果。在怀疑败血症的患者中,通常根据生理和生化参数作出决定,没有明确的内毒血症迹象。
内毒素活性(EA)的测量可以通过全血商业测定(见材料表)获得。它可以作为系统内毒血症的生物标志物,用于疾病严重程度的早期分层,特别是在有发生败血性休克8的风险的患者中。在最近出版的双盲随机对照临床试验中,该测定用于指导Polymyxin B血液灌注治疗。在危重病人中,MEDIC研究表明,EA水平增加与多器官功能障碍、重症监护病房(ICU)停留时间以及死亡率10相关。
已经开发出不同的检测检测内毒素。利穆卢斯阿莫博克莱特溶酶(LAL)测定,无论是作为凝胶凝固,浊度,或染色体检测,到目前为止一直是最经常采用的血清内毒素估计。它基于内毒素诱导马蹄蟹、利穆卢斯多聚苯酚的溶血性凝血的能力。然而,这种测定在特异性方面有一定的局限性。特别是,它也可以被内毒素以外的微生物产品激活,如真菌细胞壁的成分,并且它可以通过各种人类血浆蛋白11抑制。
在过去十年中,EA的测量被开发并验证为循环内毒血症的生物标志物。与 LAL 测试相比,EA 在临床环境中实现更快、更容易。此外,它已被证明比全血的LAL更准确,在体外和体内12的灵敏度和特异性增加。
尽管它最初作为早期诊断工具,用于快速识别 GN 细菌作为败血症致病剂,但 EA 水平也作为疾病严重程度的生物标志物进行了研究。在此背景下,它已被证明是特别有用的评估低灌注状态,由于持续的严重疾病,如败血性休克或心脏后骤停综合征13。最近,自开发净化电检系统以来,还提出了一个积极的EA结果,作为一种筛选工具,以准确识别这种治疗14的潜在候选者。我们最近对107例败血性休克患者早期高浓度EA的患病率和临床意义进行了观察性回顾性研究。根据其他最近的研究结果,我们发现EA是败血性休克15患者疾病严重程度的一个有希望的标志。
本手稿的目的是描述在床边或实验室执行 EA 测定的方法,并描述其在败血性休克的代表性情况下的潜在用途。该技术可以通过测量嗜中性粒细胞中的增强氧化性爆裂来检测LPS活性,这种活性是通过抗内毒素抗体和LPS的复合物启动的。增加的呼吸爆发由化学光度计检测,发出的光量与血液样本中的内毒素量成正比。测定需要很少的试剂,大约需要30分钟才能进行,使用全血12的40μL。
Protocol
Representative Results
Discussion
化血症休克现在仍然与死亡率高达40%有关,尽管这个比率根据考虑的报告16而变化。大多数专家都提倡需要新型和更好的生物标志物,以帮助临床医生早期诊断,更好地管理和预后化脓性休克患者6。
执行 EA 测试不需要以前的技术知识或复杂的实验室设备,任何医疗保健提供商都可以轻松快速地了解如何运行它。及时识别高循环 EA 可能有助于将?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢保罗·布拉加内和丽莎·马蒂亚斯森博士对测定协议方法的审查。达里奥·温特顿,医学博士为审查手稿的英语语言能力提供了实质性帮助。
Materials
| EAA kit | Spectral Medical Inc. | EAAST-20 | Package with 20 tests + 1 quality control |
| Smart Line TL | Berthold | EAASL | Luminometer |
| Incubator shaker | GRANT | ES-20 | Mini-incubator shaker |
| Vortexer | VWR | 444-2790 | Vortex instrument |
References
- Akira, S., Takeda, K. Toll-like receptor signalling. Nature Reviews Immunology. 4 (7), 499-511 (2004).
- Takeda, K. Evolution and integration of innate immune recognition systems: the Toll-like receptors. Journal of Endotoxin Research. 11 (1), 51-55 (2005).
- Ulevitch, R. J., Tobias, P. S. Recognition of gram-negative bacteria and endotoxin by the innate immune system. Current Opinion in Immunology. 11 (1), 19-22 (1999).
- Natanson, C., et al. Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock. The Journal of Experimental Medicine. 169 (3), 823-832 (1989).
- Suffredini, A. F., et al. The cardiovascular response of normal humans to the administration of endotoxin. The New England Journal of Medicine. 321 (5), 280-287 (1989).
- Singer, M., et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA: The Journal of the American Medical Association. 315 (8), 801-810 (2016).
- Gupta, S., et al. Culture-Negative Severe Sepsis: Nationwide Trends and Outcomes. Chest. 150 (6), 1251-1259 (2016).
- Ikeda, T., Ikeda, K., Suda, S., Ueno, T. Usefulness of the endotoxin activity assay as a biomarker to assess the severity of endotoxemia in critically ill patients. Innate Immunity. 20 (8), 881-887 (2014).
- Dellinger, R. P., et al. Effect of Targeted Polymyxin B Hemoperfusion on 28-Day Mortality in Patients With Septic Shock and Elevated Endotoxin Level. The EUPHRATES Randomized Clinical Trial. JAMA: The Journal of the American Medical Association. 320 (14), 1455-1463 (2018).
- Marshall, J. C., et al. Diagnostic and prognostic implications of endotoxemia in critical illness: results of the MEDIC study. The Journal of Infectious Diseases. 190 (3), 527-534 (2004).
- Levin, J., Bang, F. B. Clottable protein in Limulus; its localization and kinetics of its coagulation by endotoxin. Thrombosis et Diathesis Haemorrhagica. 19 (1), 186-197 (1968).
- Marshall, J. C., et al. Measurement of endotoxin activity in critically ill patients using whole blood neutrophil dependent chemiluminescence. Critical Care. 6 (4), 342-348 (2002).
- Grimaldi, D., et al. High Level of Endotoxemia Following Out-of-Hospital Cardiac Arrest Is Associated With Severity and Duration of Postcardiac Arrest Shock. Critical Care Medicine. 43 (12), 2597-2604 (2015).
- Klein, D. J., et al. The EUPHRATES trial (Evaluating the Use of Polymyxin B Hemoperfusion in a Randomized controlled trial of Adults Treated for Endotoxemia and Septic shock): study protocol for a randomized controlled trial. Trials. 15, 218 (2014).
- Bottiroli, M., et al. Prevalence and clinical significance of early high Endotoxin Activity in septic shock: An observational study. Journal of Critical Care. 41, 124-129 (2017).
- Kaukonen, K. M., Bailey, M., Suzuki, S., Pilcher, D., Bellomo, R. Mortality related to severe sepsis and septic shock among critically ill patients in Australia and New Zealand. JAMA: The Journal of the American Medical Association. 311 (13), 1308-1316 (2014).
- Biagioni, E., et al. Endotoxin activity levels as a prediction tool for risk of deterioration in patients with sepsis not admitted to the intensive care unit: a pilot observational study. Journal of Critical Care. 28 (5), 612-617 (2013).
- Roth, R. I., Levin, F. C., Levin, J. Optimization of detection of bacterial endotoxin in plasma with the Limulus test. The Journal of Laboratory and Clinical Medicine. 116 (2), 153-161 (1990).
- Yaguchi, A., Yuzawa, J., Klein, D. J., Takeda, M., Harada, T. Combining intermediate levels of the Endotoxin Activity Assay (EA) with other biomarkers in the assessment of patients with sepsis: results of an observational study. Critical Care. 16 (3), (2012).
- Earley, Z. M., et al. Burn Injury Alters the Intestinal Microbiome and Increases Gut Permeability and Bacterial Translocation. PLoS One. 10 (7), (2015).
- Munster, A. M., Smith-Meek, M., Dickerson, C., Translocation Winchurch, R. A. Incidental phenomenon or true pathology?. Annals of Surgery. 218 (3), 321 (1993).
- Clementi, A., Virzì, G. M., Brocca, A., Ronco, C. The Role of Endotoxin in the Setting of Cardiorenal Syndrome Type 5. Cardiorenal Medicine. 7 (4), 276-283 (2017).
- Virzì, G. M., et al. Cardiorenal Syndrome Type 5 in Sepsis: Role of Endotoxin. in Cell Death Pathways and Inflammation. Kidney and Blood Pressure Research. 41 (6), 1008-1015 (2016).
- Mignon, F., Piagnerelli, M., Van Nuffelen, M., Vincent, J. L. Effect of empiric antibiotic treatment on plasma endotoxin activity in septic patients. Infection. 42 (3), 521-528 (2014).
- Klein, D. J., et al. Daily variation in endotoxin levels is associated with increased organ failure in critically ill patients. Shock. 28 (5), 524-529 (2007).
