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Tecnological development of endotoxin assay with leukocyte-rich plasma and plasma in sepsis

富白细胞血浆及脓毒症血浆内毒素检测技术进展

基本信息

批准号：
22390339
负责人：
ENDO Shigeatsu
金额：
$ 11.98万
依托单位：
Iwate Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2012
项目状态：
已结题

项目摘要

For the National Health Insurance-covered endotoxin assay method for the determination of plasma endotoxin, namely, turbidimetry-gelation time assay, platelet-rich plasma (PRP) prepared by centrifugation of the patient ’s blood is used, as a rule. However, the diagnostic sensitivity of this assay can hardly be considered to be satisfactory (sensitivity, 58.6%; specificity, 97.0%). In sepsis, endotoxin recognized by leukocytes and bound to the surface of leukocytes as a bacterial cell constituent and endotoxin liberated from bacteria and bound to the CD14 receptor, toll-like receptor (TLR) 4, etc., on the leukocyte membrane surface are known; in addition endotoxin is also considered to be taken up by the cells besides being present in the circulating plasma. Endotoxin thereby activates leukocytes, which in turn, is associated with activation of humoral factors, such as cytokines. We focused our attention on the endotoxin occurring within and on the surface of white blood cells, and have reported the possibility of the sensitivity of the test being improved by simultaneously using both white blood cells and plasma as assay samples. This study was undertaken to devise and examine a new method of sample preparation for endotoxin assay in leukocyte-rich plasma (LRP), prepared taking advantage of the property of hydroxyethyl starch (HES) as an erythrocyte aggregating agent. Furthermore, we comparatively assessed the assay results obtained by the conventional method using PRP and the turbidimetry-gelation time method using LRP. The results showed significantly higher endotoxin levels in LRP, as compared to the endotoxin levels in PRP determined by the conventional method, in patients with sepsis, and a significantly higher sensitivity of the former method. We consider that our newly devised assay method may contribute to improvement of the diagnosis rate of sepsis.

按国家医保内毒素检查法测定血浆内毒素的方法，即比浊法-凝胶时间法，一般采用患者S血液离心法制备的富血小板血浆。然而，这种方法的诊断敏感性很难令人满意(敏感性，58.6%；特异性，97.0%)。在脓毒症中，内毒素被白细胞识别并作为细菌细胞成分结合到白细胞表面，从细菌中释放出来并与白细胞膜表面的CD14受体、Toll样受体(TLR)4等结合的内毒素已知；此外，内毒素除存在于循环血浆中外，还被认为被细胞摄取。因此，内毒素激活白细胞，而白细胞反过来又与体液因素的激活有关，如细胞因子。我们将注意力集中在白细胞内和表面的内毒素上，并报告了同时使用白细胞和血浆作为检测样本来提高检测灵敏度的可能性。利用羟乙基淀粉(HES)作为红细胞聚集剂的特性，设计并研究了一种新的检测富白细胞血浆(LRP)中内毒素的样品制备方法。此外，我们还比较了使用PRP的常规方法和使用LRP的浊度-凝胶时间法所获得的检测结果。结果表明，脓毒症患者LRP中的内毒素水平显著高于常规方法测定的PRP中的内毒素水平，且前者的敏感性显著高于后者。我们认为，我们新设计的检测方法可能有助于提高脓毒症的诊断率。