Medical Faculty, Division of Cardiology, Pulmonology and Vascular Medicine, University Hospital Düsseldorf, D-40225 Düsseldorf, Germany.
Free Radic Biol Med. 2013 Oct;63:236-42. doi: 10.1016/j.freeradbiomed.2013.05.018. Epub 2013 May 22.
The analytical validation of a possible biomarker is the first step in the long translational process from basic science to clinical routine. Although the chemokine-like cytokine macrophage migration inhibitory factor (MIF) has been investigated intensively in experimental approaches to various disease conditions, its transition into clinical research is just at the very beginning. Because of its presence in preformed storage pools, MIF is the first cytokine to be released under various stimulation conditions. In the first proof-of-concept studies, MIF levels correlated with the severity and outcome of various disease states. In a recent small study with acute coronary syndrome patients, elevation of MIF was described as a new factor for risk assessment. When these studies are compared, not only MIF levels in diseased patients differ, but also MIF levels in healthy control groups are inconsistent. Blood MIF concentrations in control groups vary between 0.56 and 95.6 ng/ml, corresponding to a 170-fold difference. MIF concentrations in blood were analyzed by ELISA. Other than the influence of this approach due to method-based variations, the impact of preanalytical processing on MIF concentrations is unclear and has not been systematically studied yet. Before large randomized studies are performed to determine the impact of circulating MIF on prognosis and outcome and before MIF is characterized as a diagnostic marker, an accurate protocol for the determination of reproducible MIF levels needs to be validated. In this study, the measurement of MIF in the blood of healthy volunteers was investigated focusing on the potential influence of critical preanalytical factors such as anticoagulants, storage conditions, freeze/thaw stability, hemolysis, and dilution. We show how to avoid pitfalls in the measurement of MIF and that MIF concentrations are highly susceptible to preanalytical factors. MIF serum concentrations are higher than plasma concentrations and show broader ranges. MIF concentrations are higher in samples processed with latency than in those processed directly and strongly correlate with hemoglobin in plasma. Neither storage temperature nor storage length or dilution or repeated freezing and thawing influenced MIF concentrations in plasma. Preanalytical validation of MIF is essential. In summary, we suggest using plasma and not serum samples when determining circulating MIF and avoiding hemolysis by processing samples immediately after blood drawing.
可能的生物标志物的分析验证是将基础科学转化为临床常规的漫长转化过程中的第一步。尽管趋化因子样细胞因子巨噬细胞移动抑制因子（MIF）在各种疾病条件的实验方法中得到了深入研究，但它向临床研究的转化才刚刚开始。由于其存在于预先形成的储存池中，因此 MIF 是在各种刺激条件下首先释放的细胞因子。在最初的概念验证研究中，MIF 水平与各种疾病状态的严重程度和结果相关。在最近一项急性冠状动脉综合征患者的小型研究中，MIF 的升高被描述为一种新的风险评估因素。当比较这些研究时，不仅患病患者的 MIF 水平不同，而且健康对照组的 MIF 水平也不一致。对照组血液中的 MIF 浓度在 0.56 和 95.6ng/ml 之间变化，相差 170 倍。通过 ELISA 分析血液中的 MIF 浓度。除了由于方法差异导致的这种方法的影响外，对 MIF 浓度的分析前处理的影响尚不清楚，也尚未进行系统研究。在进行大规模随机研究以确定循环 MIF 对预后和结果的影响以及将 MIF 特征化为诊断标志物之前，需要验证用于确定可重复的 MIF 水平的准确方案。在这项研究中，我们研究了健康志愿者血液中 MIF 的测量，重点关注抗凝剂、储存条件、冻融稳定性、溶血和稀释等关键分析前因素的潜在影响。我们展示了如何避免在 MIF 测量中出现陷阱，以及 MIF 浓度对分析前因素高度敏感。MIF 血清浓度高于血浆浓度，且范围更宽。用潜伏期处理的样本中的 MIF 浓度高于直接处理的样本，并且与血浆中的血红蛋白强烈相关。血浆中 MIF 浓度不受储存温度、储存时间、稀释或反复冻融的影响。MIF 的分析前验证至关重要。总之，我们建议在确定循环 MIF 时使用血浆而不是血清样本，并通过在采血后立即处理样本来避免溶血。
