从临床样本中自动分离和收集癌症相关物质
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 将 9 mL 全血收集到 EDTA 或 cfDNA 稳定的采血管中。 通过上下翻转试管约 10 次来充分混合。 将样品储存在室温（RT;用于短期储存）或4°C（用于?…
Representative Results
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?…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1% BSA (Bovine Serum Albumin) | Sigma-Aldrich | A3059 | |
| 1.5 mL Microcentrifuge Tube | Axygen | MCT-150-C-S | |
| 15 mL Conical Tube | SPL | 50015 | |
| 4150 TapeStation System | Agilent | G2992AA | Cell-free DNA Screen Tape (Agilent, 5067-5630), Cell-free DNA Sample Buffer (Agilent, 5067-5633) |
| Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit | Apostle | A17622-250 | 5 mL X 50 preps version |
| BD Vacutainer blood collection tubes | BD | 367525 | EDTA Blood Collection Tube (10 mL) |
| BioViewCCBS | Clinomics | BioView Clinomics-Customized Bioview System. Allegro Plus microscope-based customization equipment | |
| CD45 Monoclonal Antibody (HI30), PE-Alexa Fluor 610 | Invitrogen | MHCD4522 | |
| FAST Auto cartridge | Clinomics | CLX-M3001 | |
| LBx-1 cartridge | Clinomics | CLX-M4101 | |
| LBx-2 cartridge | Clinomics | CLX-M4201 | |
| OPR-2000 instrument | Clinomics | CLX-I2001 | |
| Cover Glass | Marienfeld Superior | HSU-0101040 | |
| DynaMag 2 Magnet Stand | Thermo Fisher Scientific | 12321D | |
| Ficoll Paque Solution | GE healthcare | 17-1440-03 | density gradient solution |
| Filter Tip, 10 µL | Axygen | AX-TF-10 | Pipette tips with aerosol barriers are recommended to help prevent cross contamination. |
| Filter Tip, 200 µL | Axygen | AX-TF-200 | Pipette tips with aerosol barriers are recommended to help prevent cross contamination. |
| Filter Tip, 100 µL | Axygen | AX-TF-100 | Pipette tips with aerosol barriers are recommended to help prevent cross contamination. |
| Filter Tip, 1000 µL | Axygen | AX-TF-1000 | Pipette tips with aerosol barriers are recommended to help prevent cross contamination. |
| FITC anti-human CD326 (EpCAM) Antibody | BioLegend | 324204 | |
| FITC Mouse Anti-Human Cytokeratin | BD Biosciences | 347653 | |
| Formaldehyde solution (35 wt. % in H2O) | Sigma Aldrich | 433284 | |
| Kimtech Science Wipers | Yuhan-Kimberly | 41117 | |
| Latex glove | Microflex | 63-754 | |
| Magnetic Bead Separation Rack | V&P Scientific | VP 772F2M-2 | |
| Manual Pipetting (0.5-10 µL) | Eppendorf | 3120000020 | |
| Manual Pipetting (2-20 µL) | Eppendorf | 3120000038 | |
| Manual Pipetting (10-100 µL) | Eppendorf | 3120000046 | |
| Manual Pipetting (20-200 µL) | Eppendorf | 3120000054 | |
| Manual Pipetting (100-1000 µL) | Eppendorf | 3120000062 | |
| Mounting Medium With DAPI - Aqueous, Fluoroshield | abcam | ab104139 | |
| Normal Human IgG Control | R&D Systems | 1-001-A | |
| OLYMPUS BX-UCB | Olympus | 9217316 | |
| Pan Cytokeratin Monoclonal Antibody (AE1/AE3), Alexa Fluor 488 | Invitrogen | 53-9003-82 | |
| PBS (Phosphate Buffered Saline Solution) | Corning | 21-040CVC | |
| Portable Pipet Aid | Drummond | 4-000-201 | |
| Slide Glass | Marienfeld Superior | HSU-1000612 | |
| StainTray Staining box | Simport | M920 | |
| Sterile Serological Pipette (10 mL) | SPL | 91010 | |
| Triton X-100 solution | Sigma Aldrich | 93443 | |
| TWEEN 20 | Sigma Aldrich | P7949 | |
| Whole Blood | Stored at 4-8 °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. | ||
| X-Cite 120Q (Fluorescence Lamp Illuminator) | Excelitas | 010-00157 |
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Citazione di questo articolo
Bae, J., Jeong, J., Kim, B. C., Lee, S. Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples. J. Vis. Exp. (191), e64325, doi:10.3791/64325 (2023).