Name: establishment of a clinic-based biorepository
Uploaded: 2017-05-29T14:00:00
Description: Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

这项工作得到了中西部大学健康科学研究促进委员会资助，授予EEH和中西部大学研究与研究局局长助学金，授予KJL。附属实验室和附属皮肤科提供了额外的支持。我们感谢Sarah Potekhen，Jamie Barto，Stefani Fawks，Cody Jording，Stacie Schimke，Heather Kissel和Ali Zaidi的技术援助。

所有对人体组织样品的实验均经Western IRB（WIRB Protocol＃20142461）批准。用于这些程序的组织收集的单一标准是SCC的活检证实的诊断。原始IRB方案中没有概述排除标准，但未使用已知血源性传染病患者的样本。在收集皮肤组织，血液和唾液之前获得知情同意书。中西部机构审查委员会批准了在西西里大学（AZ＃807）的基于临床的生物样品样品的验证工作。 皮肤肿瘤组织加工<p cl...

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据笔者了解，该协议是首例以成本效益和速度快的方式重点关注皮肤组织样本的临床采购。经历莫氏显微外科手术的患者通常在特定的时间段内安排，收集限于这些时期。样本收集涉及病人护理中涉及的医疗助理，处理肿瘤样本的莫氏组织学技术人员以及处理样品进入和血液处理的指定实验室工作人员的努力。 收集队的三名成员（ 即医疗助理，莫斯组织技术员和实验?...

癌症和生物标志物研究依赖于提供优质的人体组织样本，有限的供应阻碍了研究1,2 。许多皮肤病学研究受到供应不足，质量变化以及与使用人体组织有关的成本的限制。建立大型专用生物库的成本估计约为200万美元3 ，这些成本将人体组织的使用放在许多研究人员的触角之外。此外，生成和存储研究样本的过程有可能影响临床操作，并延迟患者护理，如果不慎重执行。已经建立了一个具有成本效益，基于诊所的生物反应器，其重点是按照推荐的最佳实践和样品验证4,5,6 。 <pclass =“jove_content”&gt;该协议已经在皮肤科诊所中开发，该诊所执行大量的Mohs显微手术以去除鳞状细胞癌（SCC），基底细胞癌（BCC）和黑素瘤皮肤癌。志愿者捐助者可以从这个病人群中招募。重要的是在收集部位建立生物反应器，以迅速从同意的患者中获取组织和血液，而不会延迟治疗。收集相同诊所的样品可最大限度地减少采集技术的变化，并最大限度地减少样品质量的变化，这对于下游应用7,8可能是有问题的。 莫氏显微手术技术的目的是确保所有的癌组织被去除，同时保留尽可能多的健康组织。该过程涉及逐渐去除肿瘤组织的薄层。每个连续的层是他的通过皮肤科医师进行病理学检查（在对肿瘤组织进行冷冻切片并进行H＆E染色后），以确定是否所有的癌组织都被去除。当患者留在办公室时，执行随后的组织层的切除和检查。该技术被认为是SCC 9的最佳治疗方案。此时，伤口闭合，为了改善愈合和美容外观，常常切除相邻的正常组织（ANT）。因此，这种去除肿瘤的外科手术理想地适用于收集组织学表征的组织用于将来的研究。 用于获得肿瘤组织，相邻正常组织，唾液和血液样本的采购程序被设计为对正常人员职责的影响最小（ 图1 ）。医疗助理在准备患者手术时进行抽血。在完成莫氏程序后，<html>哦，组织技术人员准备标本的附加组织学载玻片并将组织转移到生物支架。与建立生物反应器相关的成本包括购买冷冻保存冷冻机，创建适度的临床实验室空间以及开发库存跟踪计划。 <img alt="图1" src="/files/ftp_upload/55583/55583fig1.jpg" /> 图1：样本收集和负责人员的顺序。病人登记并取得病人同意后，医疗助理将收集颊拭子并进行抽血。然后皮肤科医师和医疗助理人员切除肿瘤并关闭伤口，在此期间分别收集SCC和ANT标本。专门的实验室技术人员处理血液和切片SCC和ANT标本，用于组织培养，保存和进入生物支架。ftp_upload / 55583 / 55583fig1large.jpg“target =”_ blank“&gt;请点击此处查看此图的较大版本。 收集的样品的多样性可实现多种实验方法（ 图2 ）。从患者收集的样品是颊拭子（如果需要，也可以收集唾液），全血和切除的组织。颊拭子和来自全血的样品在不加工的情况下被保存用于基因分型和组织匹配。将全血分离成白细胞（WBC）和血浆级分用于未来分析。在Mohs加工之后，将冷冻的肿瘤直接置于液氮中并转移至-80℃的冷冻器中。使用先前技术10,11的修饰培养新鲜可行的肿瘤组织和ANT样品，然后冷冻保存。在收集期间，每个样本类型的数量在进入之前记录在电子表格上<html>库存跟踪程序便于准确处理（ 表1 ）。 <img alt="图2" src="/files/ftp_upload/55583/55583fig2.jpg" /> 图2：基于临床的生物样品采集和处理的概述。从患者收集颊拭子和血液样品，并存储用于下游基因分型和组织匹配。全血进一步处理白细胞（WBC）分离和CTC分析，以及血浆采集和液体活检分析。在Mohs手术期间切除的组织进行组织学处理以用于诊断目的，之后组织学载玻片可用于实验用于进一步的免疫组织化学分析。假设切除的组织样本足够大，一部分新鲜组织被去除并切片以进行蛋白质和RNA分离，并建立培养的细胞系。55583 / 55583fig1large.jpg“target =”_ blank“&gt;请点击此处查看此图的较大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>收藏日期： </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td>病人1 </td><td>病人2 </td><td>病人3 </td><td>病人4 </td><td>病人5 </td></tr><tr><td>姓名和出生日期</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>眼睛的颜色</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>样品类型</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>样品位置</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>唾液</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> w ^洞血</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>等离子体</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>肿瘤生存</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>组织正常生存</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>肿瘤莫氏液氮</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>组织正常液氮</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>幻灯片</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表1：记录样品收集的清单。收集的每个样本跟踪和记录的数据包括患者姓名，出生日期和眼睛颜色（皮肤分类）以及位置取样。唾液样本的数量，采血量以及收集的活的和保存的组织标本的数量也被记录作为后续用途分配的参考。 <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">请点击这里下载此文件。</a> 为了验证样品收集程序，已经在下游应用中测试了每种样品类型。使用先前技术的修改12 ，肿瘤和ANT已经成功地用于蛋白质和RNA分离，并且可以潜在地用于DNA分离。已经通过显微镜评估从组织切片建立的活的外植体，而存储的组织学载玻片已被用于免疫组织化学和免疫荧光。 通过遵循这里描述的方案，可以将该模型扩展到其他皮肤科诊所，其他肿瘤类型（如黑素瘤），以及其他外科专业和实践，为人类癌症的多方面研究提供人体组织样本。对于其他实践，可能需要对该协议进行轻微的修改，但原则上该协议适用于常规地丢弃患者治疗过程中收集的患者样本的任何外科手术。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K3EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>02-685-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Double Edge Blades</td>
			<td>ThermoFisher Scientific</td>
			<td>50-949-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Medium Point General Purpose Forceps</td>
			<td>ThermoFisher Scientific</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Lonza Walkersville KGM Keratinocyte Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9791321</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron Microscopy Sciences Tissue TEK OCT Compound</td>
			<td>ThermoFisher Scientific</td>
			<td>50-363-579</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica CM1950 Cryostat</td>
			<td>Leica Biosystems</td>
			<td>14047743909</td>
			<td></td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>ThermoFisher Scientific</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Rapid-Chrome H&#38;E Frozen Section Staining Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>99-900-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Long-Term Storage Cryogenic Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>03-337-7X</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Boca Scientific BuccalT-Swab&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9679349</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Signaling Technology 10x RIPA Buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>50-195-822</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail</td>
			<td>ThermoFisher Scientific</td>
			<td>PI78443</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>05-401-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet Morter Cordless Homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>12-141-3</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Free Pellet Pestle</td>
			<td>ThermoFisher Scientific</td>
			<td>121-141-364</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma SteriCycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>13-998-089</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>SH30071.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Advanced DMEM/F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>12-634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco 1M Hepes</td>
			<td>ThermoFisher Scientific</td>
			<td>15-630-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell Culture 35 mm with Vent</td>
			<td>ThermoFisher Scientific</td>
			<td>1256591</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CFH monoclonal antibody clone OX-24</td>
			<td>Abnova</td>
			<td>MAB12583</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-p40 (p63 delta) antibody</td>
			<td>Abnova</td>
			<td>ABX-144A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti MUC-1 antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-7313</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Snail + Slug (phospho S246)</td>
			<td>Abcam</td>
			<td>Ab63568</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Phallodin</td>
			<td>Molecular Probes&#160;</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Molecular Probes&#160;</td>
			<td>P36941</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axio Imager Z1 Microscope with Axiocam camera</td>
			<td>Zeiss</td>
			<td>4300009901</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX51 phase contrast with DP72 camera</td>
			<td>Olympus</td>
			<td>IX511F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinic-based biorepository

在过去几年中，皮肤癌（ 例如鳞状细胞癌，基底细胞癌和黑素瘤）的发病率一直在增加。预计对于生物医学研究研究的皮肤肿瘤样本将会有平行的需求。然而，由于建立生物制剂的成本和缺乏可用于获得不干扰临床操作的临床样品的方案，组织的可用性是有限的。建立了一项协议，收集和处理在莫氏手术之日对常规临床手术影响最小的皮肤肿瘤和相关血液和唾液样品。 Mohs组织学技术人员从经历第一层莫氏手术的患者收集并加工肿瘤样本，用于活检证实的皮肤恶性肿瘤。在手术闭合时收集相邻的正常组织。可能收集的其他样品是全血和颊拭子。通过使用通常丢弃的组织样本，生成生物反应器，其通过基于临床和实验室设置提供了几个关键优点。这些包括广泛收集的样品;获取未确定的患者记录，包括病理报告;而对于典型的捐助者，在后续访问期间可以获得额外的样本。

肿瘤和ANT已经成功地用于蛋白质分离，并通过Western印迹进行测试。 如图3所示 ，可以在收集并存储在我们基于临床的生物反应器中的患者样品中检测到皮肤鳞状细胞癌标志物（Muc1 14 ，FHL- 115和p40 16,17 ）。 <img alt="图3" src="/files/ftp_upload/55583/55583fig3.jpg" /> <st...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

经Mohs显微手术后，皮肤肿瘤经常被丢弃。这里描述了一种方案，使临床支持人员有效地处理和存储下游实验室应用的皮肤肿瘤（ 如鳞状细胞癌，基底细胞癌和黑色素瘤）样品，而不会干扰临床操作。

建立以临床为基础的生物制剂

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

Research

JoVE Journal

Medicine

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

科研

医学

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

建立和UTI和CAUTI表征在小鼠模型

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

在兔子中使用完全的白眼切除术建立严重干眼模型

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

猪静脉移植病模型的建立与评价

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

建立简单有效的术中甲状旁腺成像大鼠模型

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

Modified Arteriovenous Fistula Surgery to Establish the RV Volume Overload Mouse Model

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model (Video) | JoVE 

出生后右心室容积超负荷小鼠模型的建立与确认

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV ...

Development of an Effective Mice Model for Studying Oronasal Fistulas

Establishment of an Oronasal Fistula Mice Model

Investigating Wound Healing in Mice Models of Oronasal Fistulas (Video) | JoVE

口鼻瘘小鼠模型的建立

This study presents a method utilizing heated ophthalmologic cautery to develop a viable model for investigating oronasal fistulas. C57BL/6 mice were used ...

A Murine Maxillary Orthodontic Model Guide to Bone Remodeling Analysis

The Establishment of a Murine Maxillary Orthodontic Model

Author Spotlight: Insights into an Efficient Murine Maxillary Orthodontic Model Protocol

Due to the lack of reproducible protocols for establishing a murine maxillary orthodontic model, we present a reliable and reproducible protocol to ...