Department of Biomedical Engineering, University of California at Irvine, Irvine, CA 92697, USA.
Lab Chip. 2018 May 1;18(9):1349-1358. doi: 10.1039/c7lc01301a.
The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence screening. The trapped single leukemia cells, e.g., THP-1, Jurkat and K562 cells, are distinguished from WBCs in the phasor-FLIM lifetime map, as they exhibit significant shift towards shorter fluorescence lifetime and a higher ratio of free/bound NADH compared to WBCs, because of their glycolysis-dominant metabolism for rapid proliferation. Based on a multiparametric scheme comparing the eight parameter-spectra of the phasor-FLIM signatures, spiked leukemia cells are quantitatively distinguished from normal WBCs with an area-under-the-curve (AUC) value of 1.00. Different leukemia cell lines are also quantitatively distinguished from each other with AUC values higher than 0.95, demonstrating high sensitivity and specificity for single cell analysis. The presented platform is the first to enable high-density size-based single-cell trapping simultaneously with RBC filtering and rapid label-free individual-leukemia-cell screening through non-invasive metabolic imaging. Compared to conventional biomolecular diagnostics techniques, phasor-FLIM based single-cell screening is label-free, cell-friendly, robust, and has the potential to screen blood in clinical volumes through parallelization.
从血液中快速筛选和分离单个白血病细胞对于早期白血病检测和肿瘤异质性研究至关重要。然而，由于白血病细胞与更丰富的白细胞（WBC）之间存在大小重叠，因此从外周血中单独分离和鉴定白血病细胞极具挑战性，通常需要免疫标记或细胞遗传学检测。在这里，我们提出了一种快速且无需标记的单个白血病细胞识别平台，该平台结合了：（1）通过单细胞捕获阵列对血细胞进行高通量基于大小的分离，以及（2）通过相矢量方法和荧光寿命成像显微镜（phasor-FLIM）对白血病细胞进行识别，以量化游离/结合烟酰胺腺嘌呤二核苷酸（NADH）之间的变化，作为活细胞代谢改变的间接测量。设计的微流控捕获阵列带有 1600 个高密度可寻址的单细胞捕获阱，可同时滤除红细胞（RBC）并捕获 WBC/白血病细胞，并且与低放大倍数成像和快速荧光筛选兼容。与 WBC 相比，从相矢量-FLIM 寿命图中可以区分出捕获的单个白血病细胞，例如 THP-1、Jurkat 和 K562 细胞，因为它们的荧光寿命明显向较短的方向移动，并且游离/结合 NADH 的比例也较高，这是因为它们的糖酵解主导的代谢可实现快速增殖。基于比较相矢量-FLIM 特征的八个参数光谱的多参数方案，通过 AUC 值为 1.00，从正常 WBC 中定量区分了掺入的白血病细胞。不同的白血病细胞系也可以彼此定量区分，AUC 值高于 0.95，表明对单细胞分析具有高灵敏度和特异性。所提出的平台是第一个能够同时进行高密度基于大小的单细胞捕获、红细胞过滤以及通过非侵入性代谢成像快速进行无标记单个白血病细胞筛选的平台。与传统的生物分子诊断技术相比，基于相矢量的单细胞筛选是无标记的、对细胞友好的、稳健的，并且具有通过并行化筛选临床体积血液的潜力。
