Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry
Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry
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DOI：
10.3791/3559
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发表时间：
2011-11-01
影响因子：
1.2
通讯作者：
Viator, John A.
中科院分区：
文献类型：
--
作者：
O'Brien, Christine M.;Rood, Kyle;Viator, John A.
循环肿瘤细胞（CTC）是从肉眼可见的肿瘤中分离出来并通过血液和淋巴系统扩散以接种继发性肿瘤的细胞（1，2，3）。CTC是转移性疾病的指标，并且它们在血液样品中的检测可用于诊断癌症和监测患者对治疗的反应。由于CTC是罕见的，在晚期癌症患者的数十亿正常血细胞中包含约一个肿瘤细胞，因此它们的检测和计数是一项艰巨的任务。我们利用大多数黑素瘤细胞中色素的存在，在定制流式细胞仪中产生光声或激光诱导的超声波，用于检测循环黑素瘤细胞（CMC）（4，5）。该过程需要使用离心分离全血样品并获得白色血细胞层。如果存在于全血中，由于密度相似，CMC将与白色血细胞分离。将这些细胞重悬于磷酸盐缓冲盐水（PBS）中并引入流量计中。不是血细胞悬浮液的连续流动，我们诱导两相流动以捕获这些细胞用于进一步研究。在两相流中，微流体系统中的两种不混溶的液体在接合处相遇并形成交替的液体段塞（6，7）。PBS悬浮的白色血细胞和空气形成用激光依次照射的微升段塞。在液相中添加表面活性剂可以形成均匀的段塞，用户可以通过改变两相的流速来产生不同尺寸的段塞。空气段塞和具有白色血细胞的PBS段塞不含光吸收剂，因此不产生光声波。然而，即使含有单个CMC的白色血细胞也会吸收激光并产生高频声波。这些产生光声波的蛞蝓被隔离并收集用于细胞化学染色以验证CMC。
Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors(1,2,3). CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient's response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)(4,5). This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid(6,7). PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.