Rosenbluth Michael J, Lam Wilbur A, Fletcher Daniel A
Department of Bioengineering, 608B Stanley Hall, Berkeley, CA 94720-3220, USA.
Lab Chip. 2008 Jul;8(7):1062-70. doi: 10.1039/b802931h. Epub 2008 Jun 5.
Pathological processes in hematologic diseases originate at the single-cell level, often making measurements on individual cells more clinically relevant than population averages from bulk analysis. For this reason, flow cytometry has been an effective tool for single-cell analysis of properties using light scattering and fluorescence labeling. However, conventional flow cytometry cannot measure cell mechanical properties, alterations of which contribute to the pathophysiology of hematologic diseases such as sepsis, diabetic retinopathy, and sickle cell anemia. Here we present a high-throughput microfluidics-based 'biophysical' flow cytometry technique that measures single-cell transit times of blood cell populations passing through in vitro capillary networks. To demonstrate clinical relevance, we use this technique to characterize biophysical changes in two model disease states in which mechanical properties of cells are thought to lead to microvascular obstruction: (i) sepsis, a process in which inflammatory mediators in the bloodstream activate neutrophils and (ii) leukostasis, an often fatal and poorly understood complication of acute leukemia. Using patient samples, we show that cell transit time through and occlusion of microfluidic channels is increased for both disease states compared to control samples, and we find that mechanical heterogeneity of blood cell populations is a better predictor of microvascular obstruction than average properties. Inflammatory mediators involved in sepsis were observed to significantly affect the shape and magnitude of the neutrophil transit time population distribution. Altered properties of leukemia cell subpopulations, rather than of the population as a whole, were found to correlate with symptoms of leukostasis in patients-a new result that may be useful for guiding leukemia therapy. By treating cells with drugs that affect the cytoskeleton, we also demonstrate that their transit times could be significantly reduced. Biophysical flow cytometry offers a low-cost and high-throughput diagnostic and drug discovery platform for hematologic diseases that affect microcirculatory flow.
血液系统疾病的病理过程起源于单细胞水平，这使得对单个细胞的测量在临床上往往比大量分析得出的总体平均值更具相关性。因此，流式细胞术一直是利用光散射和荧光标记对细胞特性进行单细胞分析的有效工具。然而，传统的流式细胞术无法测量细胞的机械特性，而细胞机械特性的改变会导致诸如败血症、糖尿病视网膜病变和镰状细胞贫血等血液系统疾病的病理生理过程。在此，我们展示了一种基于高通量微流控的“生物物理”流式细胞术技术，该技术可测量血细胞群体通过体外毛细血管网络的单细胞通过时间。为了证明其临床相关性，我们使用该技术来表征两种模型疾病状态下的生物物理变化，在这两种疾病状态中，细胞的机械特性被认为会导致微血管阻塞：（i）败血症，即血液中的炎症介质激活中性粒细胞的过程；（ii）白细胞淤滞，这是急性白血病一种通常致命且了解甚少的并发症。使用患者样本，我们发现与对照样本相比，这两种疾病状态下细胞通过微流控通道的时间和微流控通道的阻塞均增加，并且我们发现血细胞群体的机械异质性比平均特性更能预测微血管阻塞。观察到参与败血症的炎症介质会显著影响中性粒细胞通过时间群体分布的形状和幅度。发现白血病细胞亚群而非整个群体的特性改变与患者白细胞淤滞症状相关——这一全新结果可能有助于指导白血病治疗。通过用影响细胞骨架的药物处理细胞，我们还证明它们的通过时间可显著缩短。生物物理流式细胞术为影响微循环血流的血液系统疾病提供了一个低成本、高通量的诊断和药物发现平台。
