Name: automatic separation and collection of cancer-related substances from clinical samples
Uploaded: 2023-01-13T14:40:00
Description: Watch this Scientific Journal Video about Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples at JoVE.com

这份手稿得到了韩国医疗器械发展基金（KMDF，批准号RS-2020-KD000019）和韩国健康产业发展研究所（KHIDI，批准号HI19C0521020020）的部分支持。

所有全血样本均取自肺癌患者。Clinomics的研究和分析由癌症基因组学研究所进行，政府的IRB研究批准由牙山医学中心机构审查委员会（IRB NO. 2021-0802）领导，IRB编号在Clinomics注册用于研究。
1. 样品制备
<ol><li>将 9 mL 全血收集到 EDTA 或 cfDNA 稳定的采血管中。</li>
	<li>通过上下翻转试管约 10 次来充分混合。</li>
	<li>将样品储存在室温（RT;用于短期储存）或4°C（用于?...

<ol>
	<li>Babayan, A., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+liquid+biopsy+approaches+for+early+detection+and+monitoring+of+cancer.">Advances in liquid biopsy approaches for early detection and monitoring of cancer.</a> Genome Medicine. 10 (1), 21 (2018).</li><li>Crowley, E., Di Nicolantonio, F., Loupakis, F., Bardelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsy:+monitoring+cancer-genetics+in+the+blood.">Liquid biopsy: monitoring cancer-genetics in the blood.</a> Nature Reviews Clinical Oncology. 10 (8), 472-484 (2013).</li><li>Bardelli, A., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsies,+what+we+do+not+know+(yet).">Liquid biopsies, what we do not know (yet).</a> Cancer Cell. 31 (2), 172-179 (2017).</li><li>Mattox, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+liquid+biopsies+for+cancer.">Applications of liquid biopsies for cancer.</a> Science Translational Medicine. 11 (507), (2019).</li><li>Heitzer, E., Perakis, S., Geigl, J. B., Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+liquid+biopsies+for+the+early+detection+of+cancer.">The potential of liquid biopsies for the early detection of cancer.</a> NPJ Precision Oncology. 1 (1), 36 (2017).</li><li>Scudellari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myths+that+will+not+die.">Myths that will not die.</a> Nature. 582 (7582), 322-326 (2015).</li><li>Prasad, V., Fojo, T., Brada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+oncology:+origins,+optimism,+and+potential.">Precision oncology: origins, optimism, and potential.</a> The Lancet Oncology. 17 (2), 81-86 (2016).</li><li>Prasad, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspective:+The+precision-oncology+illusion.">Perspective: The precision-oncology illusion.</a> Nature. 537 (7619), 63 (2016).</li><li>Siravegna, G., Marsoni, S., Siena, S., Bardelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+liquid+biopsies+into+the+management+of+cancer.">Integrating liquid biopsies into the management of cancer.</a> Nature Reviews Clinical Oncology. 14 (9), 531-548 (2017).</li><li>Bettegowda, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+circulating+tumor+DNA+in+early-and+late-stage+human+malignancies.">Detection of circulating tumor DNA in early-and late-stage human malignancies.</a> Science Translational Medicine. 6 (224), 24 (2014).</li><li>Udomruk, S., Orrapin, S., Pruksakorn, D., Chaiyawat, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+distribution+of+cell-free+DNA+in+oncology.">Size distribution of cell-free DNA in oncology.</a> Critical Reviews in Oncology/Hematology. 166, 103455 (2021).</li><li>Paterlini-Brechot, P., Benali, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+(CTC)+detection:+clinical+impact+and+future+directions.">Circulating tumor cells (CTC) detection: clinical impact and future directions.</a> Cancer Letters. 253 (2), 180-204 (2007).</li><li>Loeian, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsy+using+the+nanotube-CTC-chip:+capture+of+invasive+CTCs+with+high+purity+using+preferential+adherence+in+breast+cancer+patients.">Liquid biopsy using the nanotube-CTC-chip: capture of invasive CTCs with high purity using preferential adherence in breast cancer patients.</a> Lab on a Chip. 19 (11), 1899-1915 (2019).</li><li>Rikkert, L. G., Van Der Pol, E., Van Leeuwen, T. G., Nieuwland, R., Coumans, F. A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugation+affects+the+purity+of+liquid+biopsy-based+tumor+biomarkers.">Centrifugation affects the purity of liquid biopsy-based tumor biomarkers.</a> Cytometry Part A. 93 (12), 1207-1212 (2018).</li><li>Sharma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cell+isolation,+culture,+and+downstream+molecular+analysis.">Circulating tumor cell isolation, culture, and downstream molecular analysis.</a> Biotechnology advances. 36 (4), 1063-1078 (2018).</li><li>Bennett, C. W., Berchem, G., Kim, Y. J., El-Khoury, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA+and+next-generation+sequencing+in+the+service+of+personalized+medicine+for+lung+cancer.">Cell-free DNA and next-generation sequencing in the service of personalized medicine for lung cancer.</a> Oncotarget. 7 (43), 71013 (2016).</li><li>Lowes, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+(CTC)+and+cell-free+DNA+(cfDNA)+workshop+2016:+scientific+opportunities+and+logistics+for+cancer+clinical+trial+incorporation.">Circulating tumor cells (CTC) and cell-free DNA (cfDNA) workshop 2016: scientific opportunities and logistics for cancer clinical trial incorporation.</a> International Journal of Molecular Sciences. 17 (9), 1505 (2016).</li><li>Bryzgunova, O. E., Konoshenko, M. Y., Laktionov, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+of+cell-free+DNA+in+different+tumor+types.">Concentration of cell-free DNA in different tumor types.</a> Expert Review of Molecular Diagnostics. 21 (1), 63-75 (2021).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumour+cells+as+an+indicator+of+early+and+systemic+recurrence+after+surgical+resection+in+pancreatic+ductal+adenocarcinoma.">Circulating tumour cells as an indicator of early and systemic recurrence after surgical resection in pancreatic ductal adenocarcinoma.</a> Scientific Reports. 11 (1), 1-12 (2021).</li><li>Heidrich, I., A&#269;kar, L., Mossahebi Mohammadi, P., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsies:+Potential+and+challenges.">Liquid biopsies: Potential and challenges.</a> International Journal of Cancer. 148 (3), 528-545 (2021).</li><li>Celec, P., Vlkov&#225;, B., Laukov&#225;, L., B&#225;b&#237;&#269;kov&#225;, J., Boor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA:+the+role+in+pathophysiology+and+as+a+biomarker+in+kidney+diseases.">Cell-free DNA: the role in pathophysiology and as a biomarker in kidney diseases.</a> Expert Reviews in Molecular Medicine. 20, 1 (2018).</li><li>Thierry, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+quantification+of+circulating+DNA+in+mice+with+human+colorectal+cancer+xenografts.">Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts.</a> Nucleic Acids Research. 38 (18), 6159-6175 (2010).</li><li>Moreira, V. G., de la Cera Mart&#237;nez, T., Gonzalez, E. G., Garcia, B. P., Menendez, F. V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+and+clearance+of+cell-free+plasma+DNA+in+hemodialysis+quantified+by+real-time+PCR.">Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR.</a> Clinical Chemistry and Laboratory Medicine (CCLM). 44 (12), 1410-1415 (2006).</li><li>Gauthier, V. J., Tyler, L. N., Mannik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+clearance+kinetics+and+liver+uptake+of+mononucleosomes+in+mice.">Blood clearance kinetics and liver uptake of mononucleosomes in mice.</a> Journal of Immunology. 156 (3), 1151-1156 (1996).</li><li>Meng, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+in+patients+with+breast+cancer+dormancy.">Circulating tumor cells in patients with breast cancer dormancy.</a> Clinical Cancer Research. 10 (24), 8152-8162 (2004).</li><li>Alix-Panabi&#232;res, C., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+circulating+tumour+cell+research.">Challenges in circulating tumour cell research.</a> Nature Reviews Cancer. 14 (9), 623-631 (2014).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+circulating+tumor+cells+in+non-small-cell-lung-cancer+patients+using+a+multi-flow+microfluidic+channel.">Isolation of circulating tumor cells in non-small-cell-lung-cancer patients using a multi-flow microfluidic channel.</a> Microsystems &amp; Nanoengineering. 5 (1), 8 (2019).</li><li>Sajay, B. N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+an+optimal+and+unbiased+approach+for+tumor+cell+isolation.">Towards an optimal and unbiased approach for tumor cell isolation.</a> Biomedical Microdevices. 15 (4), 699-709 (2013).</li><li>Bailey, P. C., Martin, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+on+CTC+biology+and+clinical+impact+emerging+from+advances+in+capture+technology.">Insights on CTC biology and clinical impact emerging from advances in capture technology.</a> Cells. 8 (6), 553 (2019).</li><li>Ahn, S. M., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+fluid+proteomics:+Prospects+for+biomarker+discovery.">Body fluid proteomics: Prospects for biomarker discovery.</a> Proteomics-Clinical Applications. 1 (9), 1004-1015 (2007).</li></ol>

cfDNA和CTC的数量和浓度取决于癌症的个体，分期和类型。这也取决于患者2，4，5，10，20的状况。特别是在癌症的早期或癌前阶段，癌症相关物质的浓度非常低，因此很有可能无法检测到。然而，早期发现对患者生存和治疗策略的建立具有非常积极的影响。由于cfDNA和CTC在血液...

早期准确地发现包括癌症在内的各种疾病是建立治疗策略的最重要因素1，2，3，4。特别是，癌症的早期发现与患者生存机会的增加密切相关5，6，7，8。最近，液体活检已成为早期发现癌症的焦点。实体瘤经历血管生成并将各种物质释放到血液中。特别是，在癌症患者的血液中发现了循环DNA（ctDNA），循环RNA（ctRNA），蛋白质，外泌体等囊泡和循环肿瘤细胞（CTC）2，9。尽管这些物质的含量存在差异，但它们不仅在早期阶段而且在后期阶段都一致观察到6，10。然而，这些个体差异非常高;例如，含有ctDNA的无细胞DNA（cfDNA）的量小于1，000ng，来自癌症患者的10 mL全血中的CTC数量小于100 11，12，13。许多研究已经使用这些含量较少的物质（即cfDNA，ctDNA和CTC）来表征癌症。为了获得准确的结果，准确分离少量高纯度13，14的物质非常重要。通常使用传统的离心方法，但根据用户的技能，它们难以处理且纯度低。自CTCs被发现以来，已经开发了几种分离技术，例如离心或密度级分离，免疫珠和微流体方法。自发现CTC以来，已经开发了几种遏制技术。然而，当需要从用于分离细胞的各种芯片和膜中分离细胞时，这些技术通常受到限制15。此外，标记方法需要FACS等设备，并且由于标记污染，下游过程存在限制。
最近，液体活检的使用有所增加，并且正在进行各种研究以早期发现癌症。虽然这种方法很简单，但在下游分析中仍然存在困难，各种研究都在试图克服这些困难16，17。此外，包括医院在内的许多场所都需要自动化、可重复和高纯度、易于使用的方法。在这里，我们开发了一种光盘实验室，用于在液体活检后自动分离血液样本中的物质。这些设备基于离心、微流体和孔径细胞捕获的原理。有三种类型的椎间盘：LBx-1 可以获取血浆和血沉棕黄层，而 LBx-2 可以从体积小于 10 mL 的全血中获取血浆和 PBMC;FAST-auto还可以使用可从光盘上去除的膜来获取CTC。每次运行最多可使用四个光盘。最重要的是，这种装置和方法的优点是它可以使用少量血液从同一样本中获得多种癌症衍生物质。这意味着患者的血液只需要抽一次。此外，它还具有排除由于血液采样时间差异而导致的错误的优点。该平台易于使用，可为液体活检和下游应用提供准确的结果。在此协议中，介绍了设备和墨盒的使用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% BSA (Bovine Serum Albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tube</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tube</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>4150 TapeStation System</td>
			<td>Agilent</td>
			<td>G2992AA</td>
			<td>Cell-free DNA Screen Tape (Agilent, 5067-5630), Cell-free DNA Sample Buffer (Agilent, 5067-5633)</td>
		</tr>
		<tr>
			<td>Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit&#160;</td>
			<td>Apostle</td>
			<td>A17622-250</td>
			<td>5 mL X 50 preps version</td>
		</tr>
		<tr>
			<td>BD Vacutainer blood collection tubes</td>
			<td>BD</td>
			<td>367525</td>
			<td>EDTA Blood Collection Tube (10 mL)</td>
		</tr>
		<tr>
			<td>BioViewCCBS</td>
			<td>Clinomics</td>
			<td></td>
			<td>BioView Clinomics-Customized Bioview System. Allegro Plus microscope-based customization equipment</td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (HI30), PE-Alexa Fluor 610</td>
			<td>Invitrogen</td>
			<td>MHCD4522</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST Auto cartridge</td>
			<td>Clinomics</td>
			<td>CLX-M3001</td>
			<td></td>
		</tr>
		<tr>
			<td>LBx-1 cartridge</td>
			<td>Clinomics</td>
			<td>CLX-M4101</td>
			<td></td>
		</tr>
		<tr>
			<td>LBx-2 cartridge</td>
			<td>Clinomics</td>
			<td>CLX-M4201</td>
			<td></td>
		</tr>
		<tr>
			<td>OPR-2000 instrument</td>
			<td>Clinomics</td>
			<td>CLX-I2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass</td>
			<td>Marienfeld Superior</td>
			<td>HSU-0101040</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag 2 Magnet Stand</td>
			<td>Thermo Fisher Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll Paque Solution</td>
			<td>GE healthcare</td>
			<td>17-1440-03</td>
			<td>density gradient solution</td>
		</tr>
		<tr>
			<td>Filter Tip, 10 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-10</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>Filter Tip, 200 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-200</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>Filter Tip, 100 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-100</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>Filter Tip, 1000 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-1000</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>FITC anti-human CD326 (EpCAM) Antibody</td>
			<td>BioLegend</td>
			<td>324204</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human Cytokeratin</td>
			<td>BD Biosciences</td>
			<td>347653</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution (35 wt. % in H2O)</td>
			<td>Sigma Aldrich</td>
			<td>433284</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech Science Wipers</td>
			<td>Yuhan-Kimberly</td>
			<td>41117</td>
			<td></td>
		</tr>
		<tr>
			<td>Latex glove</td>
			<td>Microflex</td>
			<td>63-754</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Bead Separation Rack</td>
			<td>V&#38;P Scientific</td>
			<td>VP 772F2M-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (0.5-10 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000020</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (2-20 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000038</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (10-100 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000046</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (20-200 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000054</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (100-1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000062</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI&#160;- Aqueous, Fluoroshield</td>
			<td>abcam</td>
			<td>ab104139</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Human IgG Control</td>
			<td>R&#38;D Systems</td>
			<td>1-001-A</td>
			<td></td>
		</tr>
		<tr>
			<td>OLYMPUS BX-UCB</td>
			<td>Olympus</td>
			<td>9217316</td>
			<td></td>
		</tr>
		<tr>
			<td>Pan Cytokeratin Monoclonal Antibody (AE1/AE3), Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>53-9003-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Saline Solution)</td>
			<td>Corning</td>
			<td>21-040CVC</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable Pipet Aid</td>
			<td>Drummond</td>
			<td>4-000-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Glass</td>
			<td>Marienfeld Superior</td>
			<td>HSU-1000612</td>
			<td></td>
		</tr>
		<tr>
			<td>StainTray Staining box</td>
			<td>Simport</td>
			<td>M920</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Serological Pipette (10 mL)</td>
			<td>SPL</td>
			<td>91010</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100 solution</td>
			<td>Sigma Aldrich</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN&#160;20</td>
			<td>Sigma Aldrich</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole Blood</td>
			<td></td>
			<td></td>
			<td>Stored at 4-8 &#176;C by collecting in EDTA or cfDNA stable tube : If the whole blood is insufficient in 9 mL, add PBS (phosphate buffered saline) as much as necessary.</td>
		</tr>
		<tr>
			<td>X-Cite 120Q (Fluorescence Lamp Illuminator)</td>
			<td>Excelitas</td>
			<td>010-00157</td>
			<td></td>
		</tr>
	</tbody>
</table>

automatic separation and collection of cancer-related substances from clinical samples

最近，液体活检已被用于诊断各种疾病，包括癌症。体液含有许多物质，包括来自正常组织的细胞、蛋白质和核酸，但其中一些物质也来自患病区域。体液中这些物质的调查和分析在各种疾病的诊断中起着举足轻重的作用。因此，准确分离所需物质非常重要，为此开发了几种技术。
我们开发了一种名为CD-PRIME的光盘实验室类型的设备和平台。该设备是自动化的，在样品污染和样品稳定性方面具有良好的结果。此外，它具有采集良率好、操作时间短、重现性高等优点。此外，根据要安装的椎间盘类型，可以分离含有游离DNA的血浆，循环肿瘤细胞，外周血单核细胞或血沉棕黄层。因此，体液中存在的各种材料的采集可以用于各种下游应用，包括组学研究。

该技术的目标是轻松自动地从全血中分离出癌症相关物质。特别是，任何人都可以在所有合适的研究和分析领域使用这种技术。在液体活检中，在单个血液样本中同时且可重复地分离多种物质具有重要意义。LBx-1 和 LBx-2 椎间盘用于从全血中分离血浆和血沉棕黄层或 PBMC。 图1 显示了通过应用该器件分离的材料。首先，使用LBx-1从10mL血液中获得血浆，或使用LBx-2获得PBMC。其次?...

Watch this Scientific Journal Video about Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples at JoVE.com

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

本文描述了自动化设备在轻松有效地从全血中分离和收集物质（如无细胞 DNA 和循环肿瘤细胞）的应用。

从临床样本中自动分离和收集癌症相关物质

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

我们的协议是用户友好的。使用这种技术，任何人都可以轻松地从血液中分离成分。污染可以最小化，材料可以准确分离。通过自动化和该协议的建立，研究人员可以轻松获得高纯度的癌症相关物质，这对各种下游研究很有帮助。特别是，它有望为最终用户研究提供重要优势。展示他们的程序将是癌症基因组学研究所的高级研究员兼助理经理Jay Jeong。首先，通过在仪器的触摸屏面板上选择墨盒类型来加载 LBx 1 墨盒。按箭头按钮更改样本数。然后，打开仪器门，按照墨盒支架上的编号顺序插入所有要使用的墨盒。对于总共四个墨盒，请将虚拟墨盒放在墨盒支架的空白处。使用支撑轮安装墨盒并拧紧锁紧螺母以固定它。关上门，然后按触摸板屏幕上的运行按钮。仪器关闭滤芯上的阀门，大约需要 30 秒。按照消息打开门。拆下支撑轮，然后从阀座上拆下阀门关闭的滤芯。使用血清移液器最多移取10毫升全血样品。将移液器吸头深深插入滤芯的样品入口，然后缓慢注入全血样品。将墨盒插入仪器后，关闭仪器门并按 OK 按钮。血浆和血沉棕黄层自动与全血分离，大约需要 30 分钟。一旦血浆和血沉棕黄层分开，屏幕上就会出现一条消息，并伴有警报声。通过打开门或按下停止按钮来停止警报。开门。取出墨盒并将其放在桌子上。使用一毫升移液器吸头从血浆出口回收三毫升血浆。然后，使用一毫升移液器吸头从血沉棕黄层出口回收三毫升血沉棕黄层。接下来，通过在仪器的触摸屏面板上选择墨盒名称来加载 LBx 2 墨盒。按箭头按钮更改样品数量，然后按照LBx 1小柱所示的相同步骤将小柱插入小柱支架。插入墨盒并关闭门后，按触摸板屏幕上的运行按钮。按照前面显示的步骤从阀式墨盒支架上取下阀门关闭的滤芯并将其放在桌子上。使用血清移液器移液密度梯度溶液。将移液器吸头深深插入墨盒的入口处，然后缓慢注入溶液。然后，移液全血样品并将其缓慢注入样品入口。插入并固定墨盒后，关闭盖并按 OK 按钮。一旦血浆和PBMC自动从全血中分离出来，就会出现一条消息，并伴有警报声。打开盖并卸下墨盒。使用一毫升移液器吸头从血浆出口回收三毫升血浆，然后从PBMC出口回收三毫升PBMC。同样，对于 FAST-auto 滤芯，从滤芯支架上取下阀门关闭的滤芯后，将 6 毫升 PBS 溶液注入滤芯的 PBS 入口。然后，移液三毫升从LBx 1获得的全血或血沉棕黄层，或从LBx 2获得的PBMC样品，并将样品缓慢注入样品盒的样品入口。插入墨盒并固定后，关闭门并按 OK 按钮。CTC 的自动富集完成后，停止警报。卸下墨盒并将背板拆卸器（BPR）插入墨盒正面的四个孔中。用双手拇指按压深蓝色的机翼，然后按压浅蓝色的机身，直到它发出咔嗒声。提起墨盒主体，小心地将其卸下。非常轻柔地，用镊子从边缘拿起滤膜，然后将膜放入1.5毫升管中进行核酸制备，或放在载玻片上进行染色。如有必要，使用一毫升移液器吸头将过滤后的血液回收到出血口。使用生物分析仪评估提取的cfDNA，并将cfDNA筛选带与电子梯一起使用。绿线用于对齐梯子和样品。三角形标记表示DNA条带。大多数cfDNA的长度为166个碱基对，有些以长度的整数倍存在。通常，cfDNA的浓度高达50至700个碱基对的大小。尽管从每个单独样品中获得的量存在差异，例如每毫升8.47纳克和每毫升5.2纳克，但cfDNA提取的结果在所有情况下的浓度和尺寸分布都很好。这些结果表明，LBx 1方法可以准确分离含有cfDNA的血浆。在FAST-auto中，去除膜并用荧光抗体染色以检测CTC和白细胞。放置在载玻片上的染色膜在荧光显微镜下观察。膜染色后，细胞以蓝色显示DAPI或核阳性信号，而EpCAM和CK或CTC阳性以绿色突出显示，CD45或WBC阳性以红色突出显示。去除膜并不困难。但是，有必要小心四氯化碳的损失和污染。全血，PBMC和血沉棕黄层都有。相反，您只需要设置整个音量。也可以使用其他液体，但需要进行测试。这种技术可以直接在医院操作，并且可以快速获得该物质。

Research

JoVE Journal

Cancer Research

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

乳腺癌相关的淋巴水肿的超声评价

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

科研

癌症研究

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

利用DNA染色体引发的侧群体表型，从生物样品中检测和纯化癌细胞

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

线粒体功能在癌症相关疲劳中的作用评价

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

22C3 Anti-PD-L1 抗体精矿在非小细胞肺癌活检及细胞学标本中的应用

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

肿瘤治疗胶质母细胞瘤的临床应用

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

免疫荧光对非小细胞肺癌患者循环肿瘤细胞的半自动PD-L1特征化及枚举

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

从妇科和乳腺癌肿瘤获得癌症干细胞球

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Measuring the Motor Aspect of Cancer-Related Fatigue using a Handheld Dynamometer

使用手持测功机测量癌症相关疲劳的马达方面

Cancer-related fatigue (CRF) is commonly reported by patients both during and after receiving treatment for cancer. Current CRF diagnoses rely on ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...