Jia Yanjuan, Xu Hui, Li Yonghong, Wei Chaojun, Guo Rui, Wang Fang, Wu Yu, Liu Jing, Jia Jing, Yan Junwen, Qi Xiaoming, Li Yuanting, Gao Xiaoling
1 The Institute of Clinical Research and Translational Medicine, Gansu Provincial Hospital , Lanzhou, China .
2 The Clinical Laboratory Centre, Gansu Provincial Hospital , Lanzhou, China .
Biopreserv Biobank. 2018 Apr;16(2):82-91. doi: 10.1089/bio.2017.0082. Epub 2017 Dec 12.
Although the Ficoll-Paque method is classically used to isolate peripheral blood mononuclear cells (PBMCs), modifications in this method are required for a more rapid and economic output for biobanks and clinical laboratories, particularly in developing countries. In this study, we addressed this issue by modifying the Ficoll-Paque method for the isolation of PBMCs or mononuclear cells from the peripheral and the umbilical cord blood of healthy and diseased (infected, anemic, and chronic obstructive pulmonary disease) adult individuals. In the modified method, we initiated the cell isolation process from the buffy coat layer, which appears in the interface between the plasma and sediments after centrifugation, instead of using the whole blood as described in the classic method. Although the PBMC yield by the modified method was about 12% less than in the classic method, the number of PBMCs isolated by the modified method was more than one million, which is enough for different research/diagnostic purposes, such as multi-omics detection. Assessment of cell viability and purity by hematology analyzer and trypan blue showed no significant difference between the viability and purity of the PBMCs isolated by these two methods in almost all groups, except samples from the infected and cord blood groups, where lower PBMC purity with higher granulocyte contamination were observed. In addition, at delayed processing time points, all parameters for the two methods were decreased in a time-dependent manner, especially at 8, 12, or 24 hours after the sample collection. In summary, the performance of PBMC isolation by the classic and modified methods mainly relies on the PBMC ratio in original samples. The modified method could be preferred for PBMC isolation because of its time and cost savings, especially for the biobanks and clinical laboratories in developing countries.
尽管经典的Ficoll-Paque方法用于分离外周血单个核细胞（PBMC），但为了生物样本库和临床实验室，尤其是发展中国家的实验室能更快速且经济地获得结果，需要对该方法进行改进。在本研究中，我们通过改进Ficoll-Paque方法来解决这一问题，以从健康和患病（感染、贫血和慢性阻塞性肺疾病）成年个体的外周血和脐带血中分离PBMC或单个核细胞。在改进方法中，我们从离心后血浆与沉淀物界面处出现的白膜层开始细胞分离过程，而不是像经典方法那样使用全血。尽管改进方法获得的PBMC产量比经典方法少约12%，但改进方法分离出的PBMC数量超过100万，足以满足不同的研究/诊断目的，如多组学检测。通过血液分析仪和台盼蓝评估细胞活力和纯度发现，几乎在所有组中，这两种方法分离的PBMC的活力和纯度之间没有显著差异，但在感染组和脐带血组的样本中观察到PBMC纯度较低且粒细胞污染较高。此外，在延迟处理时间点，两种方法的所有参数均呈时间依赖性下降，尤其是在样本采集后8、12或24小时。总之，经典方法和改进方法分离PBMC的性能主要取决于原始样本中的PBMC比例。改进方法因其节省时间和成本，可能更适合用于PBMC分离，特别是对于发展中国家的生物样本库和临床实验室。
