Department of Renal Medicine, Cairns Hospital, 165 Esplanade, Cairns North, Queensland 4870, Australia.
Centre for Kidney Disease Research, The University of Queensland, Brisbane, Australia.
Diagnosis (Berl). 2020 Apr 17;8(2):249-256. doi: 10.1515/dx-2019-0088. Print 2021 May 26.
Gram-negative lipopolysaccharides are potent inducers of inflammation and have been shown to be present in patients with end-stage kidney disease. There are a variety of different manufacturers and assay types to quantify endotoxin levels; however, there is no standard methodology to demonstrate its presence in plasma.
A control group consisting of haemodialysis and non-kidney disease was selected. Five sets of experiments were conducted to try and ascertain the best platform for plasma endotoxin testing. This included: testing of blank tubes; the effects of freezing, thawing and storage on recovery; the effect of different buffers; use of an endpoint assay and comparison of turbidimetric vs. chromogenic kinetic assays.
No endotoxin was detected in the blood collection tubes. Freezing and thawing per se did not affect spike recovery rates. However, the sequencing of sample dilution relative to freezing had a significant effect on endotoxin recovery. Buffers increased spike recovery at all levels of dilution. No endotoxin was demonstrated with either the turbidimetric or chromogenic kinetic assay at two different dilutions in the haemodialysis controls. The endpoint assay at a 1:5 dilution did not achieve a valid standard curve.
The findings of our study suggest that, when testing plasma samples, either a turbidimetric or chromogenic assay may be used and should be diluted with appropriate buffers to achieve optimal recovery. Studies looking to quantify the presence of plasma endotoxin need to internally validate their assays and specify their validation findings in their results.
革兰氏阴性脂多糖是强烈的炎症诱导物，已被证明存在于终末期肾病患者中。有各种不同的制造商和检测类型来定量内毒素水平；然而，目前还没有标准的方法来证明其在血浆中的存在。
选择了一组由血液透析和非肾病患者组成的对照组。进行了五组实验，试图确定用于血浆内毒素检测的最佳平台。这包括：测试空白管；冷冻、解冻和储存对内毒素回收率的影响；不同缓冲液的影响；终点测定法的使用以及浊度测定法与比色动力学测定法的比较。
在血液采集管中未检测到内毒素。冷冻和解冻本身并不影响加标回收率。然而，相对于冷冻的样品稀释顺序对内毒素回收率有显著影响。在所有稀释水平下，缓冲液均增加了加标回收率。在血液透析对照组的两种不同稀释度下，浊度测定法和比色动力学测定法均未检测到内毒素。终点测定法在 1:5 的稀释度下未达到有效的标准曲线。
本研究的结果表明，在测试血浆样本时，可以使用浊度测定法或比色动力学测定法，并且应使用适当的缓冲液进行稀释，以达到最佳的回收率。研究定量检测血浆内毒素的存在需要对内毒素检测方法进行内部验证，并在结果中明确验证结果。
