基于质谱流式的临床大队列样本一次性免疫细胞编码与分析技术研究

在临床大队列免疫细胞流式分析中，为避免批次间差异、提高数据可比性，同一批次的样本通常先编码，再一次性上机检测。典型的流式细胞术采用荧光分子标记抗体，而荧光标签数量极为有限，很难做到既编码大队列样本又能保留足够的通道检测靶标蛋白。课题拟设计一种基于质谱流式术的临床大队列编码与检测新技术。采用金属同位素浓度比例取代荧光标签标记细胞表面普遍高表达的抗原，如标记免疫细胞共同高表达的CD45——与荧光流式相比，质谱流式的标签有100多种，打破了因通道数量限制而无法同时标记大队列和检测多靶标的瓶颈。在具体案例中，采用三种不同金属（166Er、169Tm和173Yb），可以实现至少19个样本的一次性编码和检测。扩展金属数量和比例设置，可通过4-6种金属标签的编码，实现数百个临床样本的标记。该技术能大幅减少临床大样本的检测时间、降低抗体损耗、提高数据质量，为临床大队列样本的免疫细胞族群研究提供全新的手段。

For immunological flow cytometry analysis on large clinical cohort, researchers are commonly required to barcode their samples and perform the analysis simultaneously to minimize batch-to-batch variance and enhance data repeatability. Conventional flow cytometry technique adopts fluorescent tags to label target antibodies. Since the number of available fluorescent tags are limited, it is extremely challenging to barcode a big cohort and, at the same time, leave enough channels to label target signaling proteins. This project aims to develop a novel barcoding strategy for large scale clinical cohort diagnostics. Metal tags with different ratios will be applied to barcode common antigens on cell surface. For instance, CD45 is a common and highly expressed surface marker for all the immunocyte populations. Compared to fluorescence based flow cytometry, mass spectrometry based flow cytometry (also named as CyTOF) can simultaneously label more than 100 channels. This capability mitigate the bottleneck that conventional technique can hardly barcode large clinical cohort and spare enough channels for target protein detection. To demonstrate the application of this novel strategy, three metal tags namely 166Er, 169Tm and 173Yb with only 3 ratios will be able to barcode at least 19 clinical samples and allow simultaneous detection within the same batch. By increasing the metal tag number and ratio setups, researchers can easily barcode several hundred clinical samples with only 5 or 6 metal tags. The development of this technique will provide new routes for immunocyte related fundamental research.