Name: a standardized liquid biopsy preanalytical protocol for downstream circulating-free dna applications
Uploaded: 2022-09-16T13:40:00
Description: Watch this Scientific Journal Video about A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications at JoVE.com

我们要感谢癌症生物医学研究网络（CIBERONC）的支持和以下项目资助：LB CIBERONC平台：用于液体活检标准化和促进的CIBERONC平台。PI罗德里戈托莱多，（西伯利亚），2019-2021。

在提取血液样本之前，事先从参与中心获得伦理批准。以下血清和血浆分离方案是根据生物医学研究的伦理原则进行的。
注意：此处提供了开始协议之前的注意事项。在生物医学研究中使用人类样本需要事先获得伦理批准，并征得相应的知情同意。需要一个II类生物安全柜来处理血液样本。在整个手术过程中应穿戴实验室外套、防护手套和眼镜，以避免被血源性病原体感染。...

<ol>
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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=Harmonizing+cell-free+DNA+collection+and+processing+practices+through+evidence-based+guidance.">Harmonizing cell-free DNA collection and processing practices through evidence-based guidance.</a> Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 26 (13), 3104-3109 (2020).</li><li>Trigg, R. M., Martinson, L. J., Parpart-Li, S., Shaw, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+that+influence+quality+and+yield+of+circulating-free+DNA:+A+systematic+review+of+the+methodology+literature.">Factors that influence quality and yield of circulating-free DNA: A systematic review of the methodology literature.</a> Heliyon. 4 (7), 00699 (2018).</li><li>Boissier, 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=The+centrifuge+brake+impacts+neither+routine+coagulation+assays+nor+platelet+count+in+platelet-poor+plasma.">The centrifuge brake impacts neither routine coagulation assays nor platelet count in platelet-poor plasma.</a> Clinical Chemistry and Laboratory Medicine. 58 (9), 185-188 (2020).</li><li>Johansson, 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=Considerations+and+quality+controls+when+analyzing+cell-free+tumor+DNA.">Considerations and quality controls when analyzing cell-free tumor DNA.</a> Biomolecular Detection and Quantification. 17, 100078 (2019).</li><li>Casas-Arozamena, 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=Genomic+profiling+of+uterine+aspirates+and+cfDNA+as+an+integrative+liquid+biopsy+strategy+in+endometrial+cancer.">Genomic profiling of uterine aspirates and cfDNA as an integrative liquid biopsy strategy in endometrial cancer.</a> Journal of Clinical Medicine. 9 (2), 585 (2020).</li><li>Alcoceba, M., 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:+a+non-invasive+approach+for+Hodgkin+lymphoma+genotyping.">Liquid biopsy: a non-invasive approach for Hodgkin lymphoma genotyping.</a> British Journal of Haematology. 195 (4), 542-551 (2021).</li><li>Szpechcinski, A., 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=Cell-free+DNA+levels+in+plasma+of+patients+with+non-small-cell+lung+cancer+and+inflammatory+lung+disease.">Cell-free DNA levels in plasma of patients with non-small-cell lung cancer and inflammatory lung disease.</a> British Journal of Cancer. 113 (3), 476-483 (2015).</li><li>Earl, 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=Somatic+mutation+profiling+in+the+liquid+biopsy+and+clinical+analysis+of+hereditary+and+familial+pancreatic+cancer+cases+reveals+kras+negativity+and+a+longer+overall+survival.">Somatic mutation profiling in the liquid biopsy and clinical analysis of hereditary and familial pancreatic cancer cases reveals kras negativity and a longer overall survival.</a> Cancers. 13 (7), 1612 (2021).</li><li>Lampignano, 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=Multicenter+evaluation+of+circulating+cell-free+DNA+extraction+and+downstream+analyses+for+the+development+of+standardized+(pre)analytical+work+flows.">Multicenter evaluation of circulating cell-free DNA extraction and downstream analyses for the development of standardized (pre)analytical work flows.</a> Clinical Chemistry. 66 (1), 149-160 (2020).</li><li>Febbo, P. 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=Minimum+technical+data+elements+for+liquid+biopsy+data+submitted+to+public+databases.">Minimum technical data elements for liquid biopsy data submitted to public databases.</a> Clinical Pharmacology and Therapeutics. 107 (4), 730-734 (2020).</li><li>De Mattos-Arruda, L., Siravegna, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+use+liquid+biopsies+to+treat+patients+with+cancer.">How to use liquid biopsies to treat patients with cancer.</a> ESMO Open. 6 (2), 100060 (2021).</li></ol>

液体活检在癌症管理的不同时间具有许多潜在的应用。首先，在诊断时识别肿瘤分子标志物，这些标记物将表明存在潜在的肿瘤病变，可以在临床上进一步研究。其次，在治疗期间对疾病进行实时监测，评估治疗分子反应、克隆进化，以及早期发现疾病复发或治疗耐药性。最后，在手术治疗后，作为检测最小残留疾病的工具。欧洲肿瘤内科学会（ESMO）最近发布了在转移性非小细胞肺癌（NSCLC）中?...

术语“液体活检”在2010年被定义为 起源于原发肿瘤的血液和其他体液中存在分子（例如蛋白质，脱氧核糖核酸（DNA），核糖核酸（RNA）），细胞或细胞外囊泡（例如外泌体）。液体活检样本的使用彻底改变了转化肿瘤学研究，因为组织活检仅限于特定时刻的特定区域，由于肿瘤异质性，可能会错过相关的克隆。此外，液体活检在原发组织稀缺或无法获得的肿瘤类型中起着相关作用，因为它可以避免侵入性活检，从而降低患者的成本和风险。此外，肿瘤分子特征的不断发展主要是由于治疗压力，液体活检样品可以捕获肿瘤克隆动力学，因为它们可以纵向，在疾病的不同临床和治疗时间，如基线，治疗，最佳反应，疾病进展甚至之前。“实时液体活检”的概念意味着可以实时监测肿瘤的动态变化，从而允许对这种疾病进行精准医疗。液体活检在临床上具有许多潜在的应用，包括癌症的筛查和早期检测，疾病的实时监测，最小残留疾病的检测，治疗耐药性的研究机制以及治疗水平1的患者分层。在许多肿瘤类型中，早期发现疾病复发和进展是未满足的临床需求，是提高癌症患者生存率和生活质量的关键因素。常规成像方式和可溶性肿瘤标志物可能缺乏该任务所需的敏感性和/或特异性。因此，临床上迫切需要新的预测标志物，例如基于循环游离核酸的靶标物。
用于液体活检研究的样本类型包括但不限于血液、尿液、唾液和粪便样本。其他肿瘤特异性样本可以是细胞抽吸物、脑脊液、胸腔积液、囊肿和腹水液、痰液和胰液2.前一种液体可能含有不同类型的癌症衍生物质，循环肿瘤细胞（CTC），或片段，例如外泌体和无细胞循环肿瘤DNA（ctDNA）。核酸可能被包封在细胞外囊泡（EV）中，或由于细胞死亡和损伤而释放到体液中。循环游离DNA（cfDNA）主要从凋亡或坏死细胞释放到血液中，并且存在于所有个体中，在炎症或肿瘤疾病中显示水平升高3。外泌体是由含有核酸，蛋白质和脂质的细胞分泌的小细胞外囊泡（~30-150nm）。这些囊泡构成细胞间通信网络的一部分，常见于许多类型的体液中 2.封闭在EV内的核酸受到保护，免受体液中恶劣环境的影响，从而为在液体活检环境中研究这些分子提供了更强大的方法。
总体而言，液体活检样品中循环核酸的水平非常低，因此需要灵敏的检测方法，例如数字PCR或下一代测序（NGS）。样品的分析前管理对于防止血细胞裂解和完整DNA的释放至关重要，从而导致cfDNA被基因组DNA污染。此外，在提取样品时必须小心，以避免存在基于酶的分析方法的抑制剂。
在这里，我们提出了一种用于收集和储存血浆和血清样品的标准化方法，这是基于液体活检的下游应用（包括循环核酸分析）的关键第一步。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>0030 120.086</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>10 mL serological disposable pipettes</td>
			<td>BIOFIL</td>
			<td>GSP010010</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>10 mL Vacutainer K2 EDTA tube</td>
			<td>Becton Dickinson</td>
			<td>367525</td>
			<td>These tubes can be used for plasma collection</td>
		</tr>
		<tr>
			<td>15 mL polypropylene centrifuge tubes</td>
			<td>BIOFIL</td>
			<td>CFT411150</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>3.5 mL BD Vacutainer tube without anticoagulant</td>
			<td>Becton Dickinson</td>
			<td>368965</td>
			<td>Either 8.5 or 3.5 mL tubes can be used for serum collection</td>
		</tr>
		<tr>
			<td>4 mL polypropylene cryogenic vial, round bottom, self-standing</td>
			<td>Corning</td>
			<td>430662</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>4 mL Vacutainer K2 EDTA tube</td>
			<td>Becton Dickinson</td>
			<td>367864</td>
			<td>These tubes can be used for plasma collection</td>
		</tr>
		<tr>
			<td>4200 TapeStation System</td>
			<td>Agilent</td>
			<td>G2991BA</td>
			<td>Several quantification methods are available with a&#160; specific application for cfDNA</td>
		</tr>
		<tr>
			<td>5 mL serological disposable pipettes</td>
			<td>BIOFIL</td>
			<td>GSP010005</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>8.5 mL BD Vacutainer tube without anticoagulant</td>
			<td>Becton Dickinson</td>
			<td>366468</td>
			<td>Either 8.5 or 3.5 mL tubes can be used for serum collection</td>
		</tr>
		<tr>
			<td>Centrifuge, capable of ~3000 x g with a swing bucket rotor</td>
			<td>Thermo Fisher Scientific</td>
			<td>Sorvall ST 16 &#160;10688725</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>Freezer storage boxes for 1&#8211;4 mLcryogenic vials</td>
			<td>Corning</td>
			<td>431120</td>
			<td>These boxes are needed when using 4 mL vials for storage</td>
		</tr>
		<tr>
			<td>p1000 pipette tips</td>
			<td>CORNING</td>
			<td>4809</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
		<tr>
			<td>QIAamp Circulating Nucleic Acid Kit</td>
			<td>Qiagen</td>
			<td>55114</td>
			<td>Any commercially available kit that is specific for cfDNA isolation can be used with this blood prcessing protocol.</td>
		</tr>
		<tr>
			<td>Streck&#160;Cell-Free DNA BCT CE tubes 10 mL</td>
			<td>Streck</td>
			<td>218997</td>
			<td>These tubes can be used for plasma collection</td>
		</tr>
		<tr>
			<td>Temperature Freezer (-80 &#176;C)</td>
			<td>ESCO</td>
			<td>2180104</td>
			<td>Any standard tubes/equipment can be used</td>
		</tr>
	</tbody>
</table>

a standardized liquid biopsy preanalytical protocol for downstream circulating-free dna applications

术语液体活检（LB）是指来自原发性和/或转移性肿瘤的血液和其他体液中的蛋白质，DNA，RNA，细胞或细胞外囊泡等分子。LB已成为转化研究的中流砥柱，并开始成为临床肿瘤学实践的一部分，为实体活检提供了一种微创的替代方案。LB允许 通过 微创样本提取（例如血液）实时监测肿瘤。这些应用包括早期癌症检测、患者随访以检测疾病进展、评估最小残留疾病以及潜在识别分子进展和耐药机制。为了对这些可以在临床上报告的样品进行可靠的分析，应仔细考虑并严格遵守分析前程序。样品收集、质量和储存是确定其在下游应用中的有用性的关键步骤。在这里，我们介绍了液活检工作模块的标准化方案，用于收集、处理和储存血浆和血清样本，以便基于无循环DNA进行下游液体活检分析。这里介绍的协议需要标准设备，并且足够灵活，可以应用于大多数专注于生物程序的实验室。

在没有抗凝剂的情况下离心血管后，上相呈现淡黄色并对应于血清级分（图2）。该馏分被小心地除去并等分，以便随后进行分析。
溶血可能存在于血浆或血清部分，并且上相将具有红色外观，这表明溶血的存在和程度（图3）。
EDTA管离心后，几个相或层将很明显;上相（淡黄色）是血浆部分，占总血容量的55%;?...

Watch this Scientific Journal Video about A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications at JoVE.com

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

液体活检彻底改变了我们的肿瘤学转化研究方法，样本收集、质量和储存是其成功临床应用的关键步骤。在这里，我们描述了一种标准化且经过验证的下游无循环DNA应用的方案，该协议可应用于大多数转化研究实验室。

用于下游无循环DNA应用的标准化液体活检前分析方案

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

这是处理和储存血液样本的标准方案，用于基于循环游离DNA的下游液体活检应用。这是第一个发布的标准协议，尽管该技术已经使用了好几年。大多数转化或科学实验室提供的标准设备很容易遵循。该协议可由所有实验室工作人员执行。该协议有助于标准化我们收集和存储样本的方式，这将最终影响临床，并减少执行相同或相似下游分析的不同中心之间的变异性，其中精度是最重要的。这种方法在肿瘤学研究和临床应用中特别有用。然而，它也可以应用于其他非癌症疾病，其中也使用液体活检。血液样本离心后，知道要去除多少血浆或血清可能很困难，但遵循此特定指南将有助于实现这一目标。协议的其余部分非常容易遵循，实验室经验有限。演示该程序的将是来自我们实验室的技术人员豪尔赫。首先，将含有新鲜血液的试管在室温下以1600倍g离心10分钟，并施加最大休息时间。小心地从离心机中取出试管。血清上清液的上层将呈现清澈和淡黄色。检查样本是否出现溶血迹象，并在适当时记录溶血情况。在II类生物安全柜中，将血清以250微升等分试样的形式转移到收集管中。等分试样必须正确标记。立即将血清直立冷冻在零下80摄氏度的储存盒中，并记录样品储存时间。验证样品是否在所需的四小时内处理完毕。将EDTA管在室温下以1600倍g离心10分钟，并施加最大断裂。离心后，血浆上清液将呈现清澈和淡黄色。检查样品是否出现溶血迹象，并使用一次性血清移液器将血浆转移到15毫升离心管中，而不会干扰细胞层。在细胞层上方留下少量残留的血浆，大约五毫米。如果观察到溶血，请丢弃样品进行进一步分析。离心血浆和15毫升离心管，以去除从第一个离心步骤携带的任何残留的完整血细胞。小心地从离心机中取出试管，而不是管底部的细胞沉淀，并使用一次性血清移液器将一到四毫升血浆转移到一到四毫升聚丙烯低温 BIOS 或 Eppendorfs 中。必须将残留体积的血浆留在管的底部，以避免血细胞污染血浆。立即将血浆直立冷冻在零下80摄氏度的储存盒中，并记录样品储存时间。验证样品是否正确标记并在所需的四小时内处理。使用P1000和过滤尖端收集细胞层并将其转移到两毫升管中。立即冷冻并储存在零下80摄氏度。此处显示了可用于下游应用的高质量cfDNA提取示例，而此图形图像表示具有高水平基因组污染的不合适样品的示例。血浆或血清级分必须是黄色。这可能因单个样品而异。溶血的样本应丢弃。去除血浆部分时，请注意不要去除任何细胞沉淀。该协议还可用于其他基于核酸和蛋白质的应用，例如使用免疫检测技术检测血清和血浆样品中的非编码RNA或蛋白质。这是许多实验室中使用的非常标准的技术。该协议可帮助研究人员标准化我们进行分析的方式，因为这是一个关键方面，是未来临床应用所必需的。

Research

JoVE Journal

Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

科研

癌症研究

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier (MSC) for Lung Cancer Screening

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier MSC for Lung Cancer Screening

基于 rna 的液体活检: 血浆 miRNA 特征分类器 (MSC) 在肺癌筛查中的经验

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

术中高强度聚焦超声装置治疗肝转移的可行性、安全性和准确性评价

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

用于低肿瘤含量组织中肿瘤富集的自动解剖方案

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...