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

对于用于测定血浆内毒素的国民健康保险覆盖的内毒素测定方法，即浊度法-凝胶时间测定，通常使用通过离心患者血液制备的富血小板血浆（PRP）。然而，该检测的诊断灵敏度很难被认为是令人满意的（灵敏度，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，而内毒素从细菌释放并结合到CD 14受体、toll样受体（TLR）4等，在白细胞膜表面上的内毒素是已知的;此外，内毒素除了存在于循环血浆中之外，还被认为被细胞摄取。内毒素从而激活白细胞，这反过来又与体液因子如细胞因子的激活有关。我们将注意力集中在白色血细胞内和表面上的内毒素上，并报道了通过同时使用白色血细胞和血浆作为测定样品来提高检测灵敏度的可能性。本研究利用羟乙基淀粉（HES）的红细胞聚集特性，设计并考察了一种新的富白细胞血浆（LRP）内毒素测定样品制备方法。此外，我们比较评估了使用PRP的常规方法和使用LRP的浊度-凝胶时间法获得的测定结果。结果表明，与常规方法测定的PRP中的内毒素水平相比，在脓毒症患者中，LRP中的内毒素水平显著更高，并且前者方法的灵敏度显著更高。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。

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.