Translational research often involves tissue sampling and analysis. Blood is by far the most common tissue collected. Due to the many difficulties encountered with blood procurement from children, it is imperative to maximize the quality and stability of the collected samples to optimize research results. Collected blood can remain whole or be fractionated into serum, plasma, or cell concentrates such as red blood cells, leukocytes, or platelets. Serum and plasma can be used for analyte studies, including proteins, lipids, and small molecules, and as a source of cell-free nucleic acids. Cell concentrates are used in functional studies, flow cytometry, culture experiments, or as a source for cellular nucleic acids. Before initiating studies on blood, a thorough evaluation of practices that may influence analyte and/or cellular integrity is required. Thus, it is imperative that child health researchers working with human blood are aware of how experimental results can be altered by blood sampling methods, times to processing, container tubes, presence or absence of additives, shipping and storage variables, and freeze-thaw cycles. The authors of this review, in an effort to encourage and optimize translational research using blood from pediatric patients, outline best practices for blood collection, processing, shipment, and storage.
转化研究通常涉及组织采样和分析。血液是最常见的组织样本。由于从儿童身上采集血液存在许多困难，因此必须最大限度地提高采集样本的质量和稳定性，以优化研究结果。采集的血液可以保持完整，也可以分离成血清、血浆或细胞浓缩物，如红细胞、白细胞或血小板。血清和血浆可用于分析物质研究，包括蛋白质、脂质和小分子，以及作为细胞无核酸的来源。细胞浓缩物可用于功能研究、流式细胞术、培养实验或作为细胞核酸的来源。在对血液进行研究之前，需要对可能影响分析物和/或细胞完整性的实践进行彻底评估。因此，儿童健康研究人员在处理人体血液时必须意识到实验结果可能会受到血液采样方法、处理时间、容器管、添加剂的存在与否、运输和储存变量以及冻融循环的影响。 为了鼓励和优化使用儿科患者血液进行转化研究，本综述的作者们概述了血液采集、处理、运输和储存的最佳实践。
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抗凝剂，血液样本采集，DNA，乙二胺四乙酸二钠，乙酸乙二胺，核酸，血浆，RNA，转化研究，肝素
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Endotoxin contamination of blood collection tubes: an avoidable source of error
血液采集管的内毒素污染：可避免的错误来源
We read with interest the excellent review by Carolina Gillio-Meina et al.1 that covered methods for maximizing the quality and stability of blood samples in order to optimize laboratory assays. The authors discussed best practice for the collection, processing, storage, shipping, and analysis of pediatric blood samples that are precious and often difficult to obtain. Although we commend the authors for their comprehensive review, they have overlooked one important issue that can influence subsequent analyses.
我们对 Carolina Gillio-Meina 等人的优秀综述 1 感兴趣，该综述涵盖了最大化血样质量和稳定性的方法，以优化实验室分析。作者讨论了收集、处理、储存、运输和分析宝贵且常常难以获取的儿科血样的最佳实践。尽管我们赞赏作者的全面综述，但他们忽视了一个可能影响后续分析的重要问题。
Most blood collection tubes, including Becton Dickinson (BD) Vacutainers (as recommended in this article1) are variably contaminated with endotoxin.2 Endotoxin is a complex lipopolysaccharide within the outer cell membrane of Gram-negative bacteria that is readily and constantly shed into the environment, and consists of outer polysaccharide chains and an inner fatty acid region, lipid A. This lipid component has been shown to have profound immunostimulatory and inflammatory capacity and to be a particularly potent stimulator of cells of the monocyte/macrophage lineage, inducing rapid cytokine production.3 As endotoxin is highly hydrophobic and heat-resistant it is often found on plasticware routinely used in clinical and laboratory consumables, including blood collection tubes, and its presence can inadvertently lead to erroneous assays of some downstream biomarkers.4
Newhall et al. (2010) reported that biomarker assay measurements were markedly affected by endotoxin contamination when blood was incubated in various plastic lithium heparin-coated Vacutainers, whereas only low levels of endotoxin were found in glass blood collection tubes.2 In contrast, contamination of glass Vacutainers tubes, resulting in significant TNF-alpha and IL-6 production by monocytes, has also been reported.5 Due to the possibility and variability of endotoxin contamination (and the fact that many manufacturers do not guarantee blood collection tubes as endotoxin-free), both papers suggested that blood collection tubes should be tested for endotoxin before analyzing clinical samples.2, 5 Although Gillio-Meina at al. presented a comprehensive table of recommended blood collection tubes and referred to the issue of possible leukocyte activation induced by anticoagulants and the mechanics of blood sampling 1, there was no reference or discussion of endotoxin contamination.
As best practice in clinical and research settings, when endotoxin contamination is likely to affect assay outputs, it would therefore seem prudent for investigators to either (i) measure blood samples for contaminating endotoxin (LPS), (ii) use collection tubes guaranteed as 'endotoxin-free' or 'pyrogen free' (which are commercially available), or (iii) make their own 'in-house' blood collection tubes using pyrogen-free plasticware and an endotoxin-free anticoagulant. These measures will minimize erroneous assay results arising from endotoxin contamination.
1.Gillio-Meina C, Cepinskas G, Cecchini EL, Fraser DD. Translational Research in Pediatrics II: Blood Collection, Processing, Shipping, and Storage. Pediatrics. 2013;131(4):754-766.
2.Newhall KJ, Diemer GS, Leshinsky N, Kerkof K, Chute HT, Russell CB, et al. Evidence for endotoxin contamination in plastic Na+-heparin blood collection tube lots. Clin Chem. 2010;56(9):1483-1491.
3.Heine H, Rietschel ET, Ulmer AJ. The biology of endotoxin. Molecular biotechnology. 2001;19(3):279-296.
4.Gorbet MB, Sefton MV. Endotoxin: the uninvited guest. Biomaterials. 2005;26(34):6811-6817.
5.Aziz N, Irwin MR, Dickerson SS, Butch AW. Spurious tumor necrosis factor-alpha and interleukin-6 production by human monocytes from blood collected in endotoxin-contaminated vacutainer blood collection tubes. Clin Chem. 2004;50(11):2215-2216.
Conflict of Interest:
None declared