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.

中文翻译：

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通过荧光寿命成像显微镜从高密度微流控捕获阵列中快速，无标签地鉴定血液中的单个白血病细胞。

从血液中快速筛选和分离单个白血病细胞已成为早期白血病检测和肿瘤异质性研究的关键。但是，由于白血病细胞和更丰富的白细胞（WBC）之间的大小重叠，因此从外周血中单独分离和鉴定白血病细胞极具挑战性，通常需要进行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标签的单一白血病细胞鉴定平台，该平台结合了：（1）通过单细胞捕获阵列基于血容量的高通量分离血红细胞，以及（2）通过相量法和荧光寿命鉴定白血病细胞成像显微镜（相量FLIM），定量分析游离/结合烟酰胺腺嘌呤二核苷酸（NADH）之间的变化，作为间接测量活细胞代谢变化的方法。设计有1600个高度可寻址的单细胞阱的微流体阱阵列可以同时滤出红细胞（RBC）和阱WBC /白血病细胞，并且与低倍放大成像和快速荧光筛查兼容。被捕获的单个白血病细胞（例如THP-1，Jurkat和K562细胞）在相量FLIM寿命图中与WBC区别开来，因为与WBC相比，它们表现出向较短的荧光寿命和较高的自由/结合NADH比例显着转移，因为其糖酵解占主导地位的新陈代谢能快速增殖。基于比较相量FLIM签名的八个参数频谱的多参数方案，加标的白血病细胞与正常白细胞在数量上有所区别，曲线下面积（AUC）值为1.00。AUC值高于0.95的情况下，不同的白血病细胞系也可以定量区分，这表明单细胞分析具有很高的灵敏度和特异性。提出的平台是第一个能够通过无创代谢成像同时进行RBC过滤和快速无标签个体白血病细胞筛选的同时实现基于高密度大小的单细胞捕获的平台。与传统的生物分子诊断技术相比，基于相量FLIM的单细胞筛查无标签，对细胞友好，功能强大，并具有通过平行化筛查临床量血液的潜力。