Hennig Christian, Adams Nico, Hansen Gesine
Department of Paediatric Pneumology and Neonatology, Hannover Medical School, Hannover, Germany.
Cytometry A. 2009 Apr;75(4):362-70. doi: 10.1002/cyto.a.20668.
Analysis of the immense complexity of the immune system is increasingly hampered by technical limitations of current methodologies, especially for multiparameter- and functional analysis of samples containing small numbers of cells. We here present a method, which is based on the stepwise functional manipulation and analysis of living immune cells that are self-immobilized within microfluidic chips using automated epifluorescence microscopy overcoming current limitations for comprehensive immunophenotyping. Crossvalidation with flow cytometry revealed a 10-fold increased sensitivity and a comparable specificity. By using small sample volumes and cell numbers (2-10 microl, down to 20,000 cells), we were able to analyze a virtually unlimited number of intracellular and surface markers even on living immune cells. We exemplify the scientific and diagnostic potential of this method by (1) identification and phenotyping of rare cells, (2) comprehensive analysis of very limited sample volume, and (3) deep immunophenotyping of human B-cells after in vitro differentiation. Finally, we propose an informatic model for annotation and comparison of cytometric data by using an ontology-based approach. The chip-based cytometry introduced here turned out to be a very useful tool to enable a stepwise exploration of precious, small cell-containing samples with an virtually unlimited number of surface- and intracellular markers.
当前方法的技术局限性日益阻碍了对免疫系统巨大复杂性的分析，尤其是对于含有少量细胞的样本进行多参数和功能分析时。我们在此提出一种方法，该方法基于对活免疫细胞的逐步功能操作和分析，这些细胞利用自动落射荧光显微镜在微流控芯片内实现自我固定，克服了当前全面免疫表型分析的局限性。与流式细胞术的交叉验证显示灵敏度提高了10倍，特异性相当。通过使用少量样本体积和细胞数量（2 - 10微升，低至20,000个细胞），我们甚至能够对活免疫细胞上几乎无限数量的细胞内和表面标志物进行分析。我们通过以下方式举例说明了该方法的科学和诊断潜力：（1）鉴定稀有细胞并进行表型分析，（2）对非常有限的样本体积进行全面分析，（3）对体外分化后的人B细胞进行深度免疫表型分析。最后，我们提出了一种基于本体论方法的信息学模型，用于注释和比较细胞计数数据。这里介绍的基于芯片的细胞术被证明是一种非常有用的工具，能够对珍贵的、含有少量细胞的样本进行逐步探索，同时分析几乎无限数量的表面和细胞内标志物。
