Continuous flow microfluidic device for rapid erythrocyte lysis
Continuous flow microfluidic device for rapid erythrocyte lysis
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DOI：
10.1021/ac049429p
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发表时间：
2004-11-01
影响因子：
7.4
通讯作者：
Toner, M
中科院分区：
文献类型：
--
作者：
Sethu, P;Anahtar, M;Toner, M
从全血中分离白细胞以研究炎症需要去除污染的红细胞。然而，白细胞对长时间暴露于高/低渗溶液、温度变化、机械操作和梯度离心敏感。尽管在红细胞裂解过程中要注意尽量减少白细胞活化和细胞损失，但通常不可能完全避免。大多数从白细胞制剂中去除污染红细胞的程序都是为批量处理血液而设计的，其中样品操作的时间比单细胞水平所需的时间更长。氯化铵介导的裂解是最常用的方法，以获得丰富的白细胞群体，但已被证明会导致一些激活和某些细胞类型的选择性损失。NH（4）Cl介导的裂解后，白细胞产量和随后残留白细胞的活化状态取决于暴露于裂解缓冲液的时间。我们已经开发了一种微流体裂解装置，处理红细胞去除在几乎单细胞水平。我们可以实现红细胞的完全裂解和类似于100%的白细胞回收，其中细胞暴露于等渗裂解缓冲液少于40 s，之后白细胞立即返回到生理条件。从理论上讲，这个过程可以大规模并行处理几毫升全血，在不到15分钟的时间内获得纯白细胞群。
Leukocyte isolation from whole blood to study inflammation requires the removal of contaminating erythrocytes. Leukocytes, however, are sensitive to prolonged exposure to hyper/hypoosmotic solutions, temperature changes, mechanical manipulation, and gradient centrifugation. Even though care is taken to minimize leukocyte activation and cell loss during erythrocyte lysis, it is often not possible to completely avoid it. Most procedures for removal of contaminating erythrocytes from leukocyte preparations are designed for bulk processing of blood, where the sample is manipulated for longer periods of time than necessary at the single-cell level. Ammonium chloride-mediated lysis is the most commonly used method to obtain enriched leukocyte populations but has been shown to cause some activation and selective loss of certain cell types. The leukocyte yield and subsequent activation status of residual leukocytes after NH(4)Cl-mediated lysis have been shown to depend on the time of exposure to the lysis buffer. We have developed a microfluidic lysis device that deals with erythrocyte removal at nearly the single-cell level. We can achieve complete lysis of erythrocytes and similar to100% recovery of leukocytes where the cells are exposed to an isotonic lysis buffer for less than 40 s, after which the leukocytes are immediately returned to physiological conditions. Theoretically, this process can be made massively parallel to process several milliliterss of whole blood to obtain a pure leukocyte population in less than 15 min.