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jmxnqred6

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It is generally accepted that most cancer-related mortalities
result from metastatic disease.1 While the process of metastasis
is not well understood, a number of chemical, physical, and
molecular events occur that ultimately result in the dissemination
and deposition of tumor cells into targeted organs using the
circulatory system and/or bone marrow as the carrier(s). This
“seed and soil” process was proposed as early as 1889 by Paget
and was later modified with the caveat that shed tumor cells
consist of a heterogeneous population with subpopulations
possessing different metastatic potentials. The fundamental
entities primarily responsible for spawning metastatic disease
are circulating tumor cells (CTCs), which can be produced
during early stages of tumorigenesis.4 Elucidating the quantity
of CTCs in peripheral blood or bone marrow can serve as an
indicator for the clinical management of several cancer-related
diseases by providing information on the success/failure of
therapeutic intervention and disease stage forecasting.5, The
isolation and enumeration of exfoliated CTCs in peripheral blood
or bone marrow for a variety of cancer-related diseases has
already been reported for a variety of cancer-related diseases,
such as breast,79 colorectal,10 prostate,11 renal,12 bladder,13 and
nonsmall cell lung14 cancers. As an example of the clinical utility
of CTC information, Cristofanilli et al. recently reported a study
of 177 breast cancer patients using the amount of CTCs in
peripheral blood as an indicator of survival.7 Patients with g5
CTCs per 7.5 mL of whole blood possessed a median progression-
free survival of 2.7 months versus 7.0 months for those
patients containing <5 CTCs in 7.5 mL of their peripheral blood.
The major issue with securing viable clinical information via
quantification of CTC levels is the extremely low abundance
or rare-event nature of these cells among a high number of
† Department of Chemistry, Louisiana State University.
‡ Department of Mechanical Engineering, Louisiana State University.
§ Center for BioModular Multi-scale Systems, Louisiana State University.
| Center for Advanced Microstructures and Devices, Louisiana State
University.
⊥ Department of Bioengineering, Louisiana Tech University.
(1) Leaf, C. Fortune 2004, 76–94.
(2) Paget, S. Lancet 1889, 1, 571.
(3) Fidler, I. J.; Yano, S.; Zhang, R. D.; Fujimaki, T.; Bucana, C. D. Lancet
Oncol. 2002, 3, 53–57.
(4) Loberg, R. D.; Fridman, Y.; Pienta, B. A.; Keller, E. T.; McCauley,
L. K.; Taichman, R. S.; Pienta, K. J. Neoplasia 2004, 6, 302–309.
(5) Braun, S.; Marth, C. New Engl. J. Med. 2004, 351, 824–6.
(6) Singletary, S. E.; Connolly, J. L. Ca-Cancer J. Clin. 2006, 56, 37–
47.
Published on Web 06/17/2008
10.1021/ja8015022 CCC: $40.75  2008 American Chemical Society J. AM. CHEM. SOC. 2008, 130, 8633–8641 9 8633
spectator cells in peripheral blood.6,15–18 For example, it is
clinically useful to quantitatively enumerate 0-10 CTCs in
whole blood composed of >109 erythrocytes and >106 leukocytes
per mL.7
For sampling rare events in a large population, three important
metrics must be assessed: (1) throughput, the number of cell
identification or sorting steps per unit time; (2) recovery, an
indicator of the fraction of target cells collected from the input
sample; and (3) purity, which depends on the number of
“interfering” cells excluded from the analysis.19 In addition to
these three metrics, highly efficient quantification of the number
of enriched cells must be provided as well.
The approaches used to date to enrich CTCs from clinical
samples have provided lower-than-desired recoveries with high
purity, relatively poor purity but with high recoveries, or, in
other cases, highly specialized sample processing and handling
whose success is laboratory dependent.20–25 For example,
immunomagnetic approaches for CTC enrichment using ferromagnetic
micrometer-sized particles coated with molecular
recognition elements specific for antigenic-bearing target cells
can interrogate diluted blood samples typically yield modest
recoveries (∼70%) but extremely favorable purity.22 In the case
of size-based separations employing nuclear tracked membranes,
polycarbonate membranes with varying pore sizes (8-14 μm)
can filter large volumes (9.0-18 mL) of blood and recover
nearly 85% of the CTCs, but significant numbers of leukocytes
are also retained (i.e., low purity) potentially complicating the
enumeration process.26 Investigations utilizing reverse-transcription
PCR, in which mRNAs are used as surrogates to report
CTC levels, have the ability to detect one CTC in an excess of
106 mononucleated cells.27 However, these assays are prone to
high interlaboratory variability and require extensive sample
handling and manipulation.
Most of the CTC isolation/sorting tools currently in use
possess some common procedural characteristics that make them
prohibitively difficult to implement, such as the ability to sort
only the mononucleated fraction of whole blood requiring
density gradient centrifugation prior to enrichment and the use
of either flow cytometry or fluorescence microscopy following
cell staining to enumerate the enriched CTCs. These additional
steps require sample handling and transfer, which can induce
cell loss or contamination that can dramatically affect the assay
result, especially when dealing with low numbers of targets.
Microfluidics provides a venue for producing integrated
systems that can process clinical samples in closed architectures
to minimize sample contamination and loss. However, high
throughput sampling of relatively large volumes (>1 mL) has
not been a mainstay for microfluidics due to the macro-to-micro
dilemma resulting from the small dimensional features associated
with these devices. For example, exhaustively sampling a
1.0 mL volume input using a microchannel of 30 μm × 30 μm
at a linear velocity of 1.0 mm s-1 would require 309 h (12.9
days). This sampling bottleneck was recently addressed by
studies with a glass-based microfluidic device fabricated using
deep reactive ion etching.28 The high-surface area immunological
capture bed consisted of microposts (100 μm diameter × 100 μm tall) arranged in an equilateral triangular format; the
device was capable of capturing ∼60% of CTCs from untreated
whole blood with enumeration achieved by cell staining and
microscopic visualization.
Herein we report our efforts aimed toward the development
of a self-contained system capable of meticulously separating
intact CTCs from peripheral blood and directly quantifying the
CTC level upon isolation and enrichment. As a result of careful
fluidic design rules and system integration methods, we have
successfully employed a microfluidic system capable of exhaustively
and rapidly interrogating >1.0 mL of unprocessed
whole blood possibly harboring low-abundant CTCs. At the
heart of the system were carefully engineered, exceedingly
efficient high-aspect ratio capture beds decorated with mABs
specific for antigenic integral membrane proteins expressed in
CTCs of epithelial origin and a label-free, highly specific singlecell
conductivity sensor. The device operational characteristics
were achieved by tailoring the dominant CTC capture dynamics,
specific device architecture, and suspension linear velocity in a
high throughput microsampling unit (HTMSU) containing highaspect
ratio microstructures replicated in a polymeric substrate.
Direct single-cell counting of the captured cells was made
possible by their release as a result of enzymatic digestion of
cell-antigen/antibody-surface complexes. Quantitative assessment
of CTC numbers was accomplished using an integrated
conductivity sensor capable of specifically detecting CTCs via
their electrical signatures without requiring cell staining or
microscopic visualization.

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1楼2011-01-05 22:55:03

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jmxnqredlds

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ringzhu(金币+2):fantastic~~ 2011-01-06 17:01:13

人们普遍认为大多数癌症导致的死亡源于疾病位置的转移，虽然疾病转移的过程并没有得到很好的了解，但一些生物体内化学、物理、分子反应最终导致肿瘤细胞通过循环系统或者以骨髓为载体进入完整器官而实现传播和积累沉淀。这种“种子和土壤”过程理论最早由佩吉特提出，后又进行过修改，提出不要让那些存在于不同种类的细胞群中具有不同转移潜能的分类肿瘤细胞流动的警告。负责“做种”转移疾病任务最基本的实体主要是CTC,它在肿瘤发生初期就可以产生。对在周边血液或骨髓的CTC数量进行说明，对于几种与癌症相关的疾病的临床管理通过提供在治疗干预和疾病阶段预测的成功或失败的信息可能起预防作用。已报道在一系列与癌症相关的疾病如：乳腺癌、结肠癌、前列腺癌、肾癌、膀胱癌以及非小细胞肺癌，可以从外围血液或者骨髓内分离和计数剥落的CTC。例如在CTC信息的临床应用中，Cristofanilli et al.最近利用从177名乳腺癌患者外围血液中测得的CTC数量作为预测她们存活下来的指示器。那些每7.5毫升血液中超过5个CTC细胞的患者平均存活2.7个月，而那些7.5毫升血液中少于5个CTC的患者则平均存活7.0个月。
通过CTC水平的量化来获取可实行的临床信息的主要争论点是在周边血液高数量的其他细胞中这类细胞极其稀少。例如：在临床上，每一毫升血液中定量计数存在0－10个CTC，而其中也包含>109个红细胞>106白细胞。
在大量细胞群中进行对稀少细胞的抽样检测，三个重要的指标需要评估：1，通过量，每一单位时间鉴定或拣选的细胞数量；2，回收率，输入样品中靶细胞的收集程度；3，纯度指数，取决于在分析中排除的干扰细胞的数量。除了这三个指标以外，也必须提供富集细胞达到的高计量效率
迄今被用于从临床样品中富集CTCs的方法，或者是纯度高而回收率达不到要求，又或者是低纯度但又具有很高的回收率，在其他情况下，实验室则依赖于高专业性的样品加工和处理的成功。例如，利用磁免疫方法进行CTC的富集中，应用具有磁性微米大小包裹着可识别的分子的粒子定向作用于反抗原靶细胞，这种方法能够对稀释的血液样品可以达到中等回收率(∼70%) 而又有极好的纯度指数。基于大小分离的方法中应用细胞穿过膜，聚碳酸酯膜具有多种不同种类大小毛孔(8-14 μm)，能够对大容量(9.0-18 mL)血液进行过滤处理，而其CTC的回收率可以达到85％，但是一定数量的白细胞也被滞留在上面，从而降低了纯度，而阻碍了分离过程。调查研究利用反转录酶链反应，mRNAs可以作为预测CTC数量水平的标志，这种方法可以从超过106个单核细胞中检测到一个CTC，但是这种方法倾向于实验室之间高度可变性，并且要求具有很高的样品操作和处理。
大多数CTC的分离和提纯工具目前通常是应用一些相同的程序化特点，这就使得它们望而却步难以实施，例如仅仅能够处理血液中单核细胞片断的特性倾向于使用密度梯度离心手段进行浓缩，而应用流体式细胞仪或荧光显微镜追踪着色细胞来对富集CTC进行计数。这些附加的步骤需要样本的处理和转移，这些都能会导致细胞的损坏和样品的污染，从而对分析结果有重大影响，特别是当处理低数量靶细胞时。
微流体提供了一个完整系统，它能够在一个很紧密的空间内进行样品检测，从而减少样品的污染和损失。但是，相应的高通量(>1 mL)的试样并没有广泛应用于微流体中，这是因为与设备相关联的很小的空间特性导致其小而又小的被选用。例如，即使极尽全力用一条30 μm × 30 μm 的微通道对1.0 mL的试样以1.0 mm s-1的线速度进行检测也需用时309 h (12.9 天)。近来这种进样瓶颈通过研究深反应离子蚀刻技术制得的玻璃微流体设备而得到解决。这种高表面积免疫捕捉壁由设计成等边三角形格式的微柱(直径100 μm × 高100 μm )组成。这种设备能够从未治疗过的血液中捕捉到接近60%的CTC，同时可以通过细胞着色和显微观察技术成功对其进行计数。
在这里我们报道出我们在关于发展这一独立系统所作出的努力，它能够小心翼翼地从外围血液中分离完整CTC并直接在分离和富集地基础上对其CTC水平进行定量测定。由于遵循严谨的流体设计规则和完整系统方法，我们已成功的应用了一个微流体系统，它能够尽量并快速的对>1.0 mL 的未处理血液进行检测，可以检测出低量的CTC。这个系统的重点已被细心规划，极其有效的高捕捉效率壁由mABs 修饰，能够特异性的结合源于上皮组织的CTC中表达的的抗原整合膜蛋白质，壁上还有无标记高效特异性单细胞传导率感应器。在复制聚合物层得到的具有高比率微观结构的高通量微样检测单元(HTMSU)中，这种装置的运行成功整合了占主导地位的CTC捕捉动力技术、特殊的设备结构和悬浮线速度。再通过酶对细胞－抗原或抗体－表面化合物进行分解从而释放出细胞，便可以直接对捕捉到的单细胞进行计数。利用一块完整的电导率传感器可以不经过细胞着色和显微观察的方法特异性地察觉CTC上的电感信号，从而完成对CTC数目的定量估计。

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3楼2011-01-06 16:16:12

已阅关注TA 给TA发消息送TA红花 TA的回帖

slz146

木虫 (正式写手)

引用回帖:

Originally posted by jmxnqredlds at 2011-01-06 16:16:12:
人们普遍认为大多数癌症导致的死亡源于疾病位置的转移，虽然疾病转移的过程并没有得到很好的了解，但一些生物体内化学、物理、分子反应最终导致肿瘤细胞通过循环系统或者以骨髓为载体进入完整器官而实现传播和积累 ...

感觉翻译的还不错啊，咋没给金币的

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4楼2011-01-27 09:58:18

已阅关注TA 给TA发消息送TA红花 TA的回帖