Gaudillière Brice, Ganio Edward A, Tingle Martha, Lancero Hope L, Fragiadakis Gabriela K, Baca Quentin J, Aghaeepour Nima, Wong Ronald J, Quaintance Cele, El-Sayed Yasser Y, Shaw Gary M, Lewis David B, Stevenson David K, Nolan Garry P, Angst Martin S
Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford University, School of Medicine, Stanford, California, 94305.
Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, California, 94305.
Cytometry A. 2015 Sep;87(9):817-29. doi: 10.1002/cyto.a.22720. Epub 2015 Jul 17.
Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients' circulating immune system. As interest in the "bedside" application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL(-1)) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort.
单细胞技术在揭示驱动人类疾病的分子和生物学过程方面具有巨大潜力。质谱流式细胞术（或飞行时间质谱流式细胞术，CyTOF）已被应用于临床研究，以全面检测患者的循环免疫系统。随着对质谱流式细胞术“床边”应用的兴趣日益增长，需要明确相关的方法学问题。本报告使用一个新生成的数据集来讨论在临床研究中实施质谱流式细胞术时的重要方法学考虑因素。具体而言，讨论了全血样本与外周血单核细胞（PBMC）的使用、质量标记抗体面板的设计、样本条形码的技术和分析意义，以及传统和无监督方法在分析高维质谱流式细胞术数据集方面的应用。在一项对19名有足月或早产史的女性进行的横断面研究中实施了质谱流式细胞术检测，以确定在非妊娠状态下外周血中的免疫特征是否能区分这两组。在用脂多糖（0、0.1、1、10和100 ng mL(-1)的LPS）刺激的全血样本中同时分析了27种表型和11种细胞内标志物，以检查Toll样受体4（TLR4）途径内的剂量依赖性信号反应。基于免疫细胞亚群的传统或无监督门控策略的补充分析表明，早产史女性的经典单核细胞中的prpS6和pMAPKAPK2反应增强（FDR<1%）。结果表明，易早产的女性在怀孕期间可能更容易出现加剧的TLR4反应。这一重要的产生假设的发现表明了单细胞质谱流式细胞术在相对较小的患者队列中检测生物学上重要差异的能力。
