Shah Jaynish S, Soon Patsy S, Marsh Deborah J
Hormones and Cancer Group, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, St. Leonards, New South Wales, Australia.
South Western Sydney Clinical School, University of New South Wales, Kensington, New South Wales, Australia.
PLoS One. 2016 Apr 7;11(4):e0153200. doi: 10.1371/journal.pone.0153200. eCollection 2016.
microRNAs have emerged as powerful regulators of many biological processes, and their expression in many cancer tissues has been shown to correlate with clinical parameters such as cancer type and prognosis. Present in a variety of biological fluids, microRNAs have been described as a 'gold mine' of potential noninvasive biomarkers. Release of microRNA content of blood cells upon hemolysis dramatically alters the microRNA profile in blood, potentially affecting levels of a significant number of proposed biomarker microRNAs and, consequently, accuracy of serum or plasma-based tests. Several methods to detect low levels of hemolysis have been proposed; however, a direct comparison assessing their sensitivities is currently lacking. In this study, we evaluated the sensitivities of four methods to detect hemolysis in serum (listed in the order of sensitivity): measurement of hemoglobin using a Coulter® AcT diff™ Analyzer, visual inspection, the absorbance of hemoglobin measured by spectrophotometry at 414 nm and the ratio of red blood cell-enriched miR-451a to the reference microRNA miR-23a-3p. The miR ratio detected hemolysis down to approximately 0.001%, whereas the Coulter® AcT diff™ Analyzer was unable to detect hemolysis lower than 1%. The spectrophotometric method could detect down to 0.004% hemolysis, and correlated with the miR ratio. Analysis of hemolysis in a cohort of 86 serum samples from cancer patients and healthy controls showed that 31 of 86 (36%) were predicted by the miR ratio to be hemolyzed, whereas only 8 of these samples (9%) showed visible pink discoloration. Using receiver operator characteristic (ROC) analyses, we identified absorbance cutoffs of 0.072 and 0.3 that could identify samples with low and high levels of hemolysis, respectively. Overall, this study will assist researchers in the selection of appropriate methodologies to test for hemolysis in serum samples prior to quantifying expression of microRNAs.
微小RNA已成为许多生物学过程的强大调节因子，并且它们在许多癌症组织中的表达已显示与癌症类型和预后等临床参数相关。微小RNA存在于多种生物体液中，被描述为潜在的非侵入性生物标志物的“金矿”。溶血时血细胞中微小RNA含量的释放会显著改变血液中的微小RNA谱，可能影响大量提议的生物标志物微小RNA的水平，从而影响基于血清或血浆检测的准确性。已经提出了几种检测低水平溶血的方法；然而，目前缺乏对它们敏感性的直接比较评估。在本研究中，我们评估了四种检测血清中溶血的方法的敏感性（按敏感性顺序列出）：使用库尔特®AcT diff™分析仪测量血红蛋白、目视检查、通过分光光度法在414nm处测量血红蛋白的吸光度以及富含红细胞的miR-451a与参考微小RNA miR-23a-3p的比率。miR比率可检测到低至约0.001%的溶血，而库尔特®AcT diff™分析仪无法检测到低于1%的溶血。分光光度法可检测到低至0.004%的溶血，并与miR比率相关。对86份来自癌症患者和健康对照的血清样本进行溶血分析表明，86份样本中有31份（36%）通过miR比率预测为溶血，而这些样本中只有8份（9%）呈现可见的粉红色变色。使用受试者工作特征（ROC）分析，我们确定了吸光度截止值分别为0.072和0.3，可分别识别低水平和高水平溶血的样本。总体而言，本研究将帮助研究人员在定量微小RNA表达之前选择合适的方法来检测血清样本中的溶血情况。
