我们感谢医院和病人的所有医务和辅助人员同意参加。我们感谢加布里埃尔白狼, 博士, 从 Edanz 集团 (www.edanzediting.com/ac) 编辑本报告草稿。这项研究得到了来自山梨州 (Y.H. 和作案手法) 的基因组研究项目的资助, 以及 YASUDA 医学基金会 (Y.H.) 的赠款。

1. 利用普通玻片进行快速显微评估的 TIC 准备
<ol><li>在临床病理组织材料可用后, 尽快完成 TIC 的准备工作。如果 TIC 标本不能立即准备好, 保持用盐水湿不育纱布覆盖的组织材料, 并贮存在冰箱中, 以防止组织的干燥。</li>
	<li>准备5毫米3组织材料, 如实体肿瘤 (如, 肝, 肺和乳腺组织), 临床上通过手术或内窥镜获得。<ol>
<li>如果组织表面有大量血液, 用无菌纱布涂上生理盐水并清除血液, 轻轻擦拭组织。</li>
		<li>对于显微标本如活检材料, 保持样品湿润与消毒纱布浸泡在生理盐水。</li>
	</ol>
</li>
	<li>切除和修剪正常组织与修剪刀和暴露表面的肿瘤病变, 如果肿块是不可见的严重。</li>
	<li>用戴手套的手将切除标本的肿瘤表面接触到正常的玻璃滑梯上几次。直观地确认触摸区域是正常玻璃滑块的80% 以上。</li>
	<li>轻轻按普通玻璃滑动对聚乙烯乙二醇 (笔) 膜滑动和摩擦轻轻 2-3 倍戴手套的手。目视确认细胞从正常的玻璃滑块转移到笔膜滑动。</li>
	<li>空气干燥两个玻璃和笔膜在室温下滑动5分钟。</li>
	<li>染色正常的玻璃滑块直接细胞学检查。用固定液蘸5秒的玻璃滑块, 然后用姬姆萨染色溶液染色十五年代。</li>
	<li>用显微镜检查和筛查正常玻璃上的肿瘤含量和细胞构成, 并进行快速评估。根据多种标准对肿瘤细胞进行评价;核扩张, 异常核型, 丰富的染色质, 细胞分布不均, 核组分/细胞质组分的比例, 细胞大小和细胞极性。</li>
</ol>2. 用于基因检测的笔膜滑动膜的制备
<ol><li>如果样本显示肿瘤细胞构成超过60% 通过快速显微评估 (步骤 1.8), 削减了用刀和戴手套的手 DNA 提取的笔膜幻灯片的肿瘤接触膜。用 pincette 和戴手套的手将切割膜转移到不育的离心管上。</li>
	<li>如果肿瘤细胞构成通过快速显微评估 (步骤 1.8) 确定为低 (小于60% 的肿瘤含量), 则使用激光捕获显微切割和获得肿瘤样本。<ol>
<li>执行姬姆萨染色, 以评估肿瘤细胞使用标准协议。</li>
		<li>通过适当的激光捕获显微切割, 切下膜的 PEM 膜滑动。</li>
		<li>用 pincette 和戴手套的手将切割膜转移到不育的离心管上。</li>
		<li>将含膜的离心管存放在4摄氏度, 直到 DNA 提取 (该协议可以在此暂停)。</li>
	</ol>
</li>
</ol>3. DNA 提取
<ol><li>根据制造商的指示进行小的修改, 使用 FFPE dna 提取试剂盒从 TIC 或 FFPE 组织样品中提取 dna。FFPE DNA 提取步骤中有一个等价的套件。</li>
	<li>添加180µL 组织裂解缓冲液 (pH = 8.3) 到含膜离心管与手动200µL 吸管。添加20µL 的蛋白酶 K 与手动20µL 吸管和混合涡流与漩涡搅拌机在最大速度 (约 2500 rpm) 5 s。</li>
	<li>用空气孵化器在一夜之间孵化56摄氏度的样品。</li>
	<li>分别在热块中孵育90摄氏度的 FFPE 和 TIC 样品1小时和10分钟。在室温下用微型离心机将离心管在 1500 x g 处旋转5秒。</li>
	<li>用200µL 的吸管将200µL 的溶解缓冲液添加到样品中, 并在5秒内以最大速度混合涡流。</li>
	<li>添加200µL 乙醇 (96-100%) 与200µL 的吸管, 并彻底混合涡流在最大速度 5 s. 在室温下, 用迷你离心机在 1500 x g 处向下旋转离心管, 其 5 s。</li>
	<li>小心地将整个裂解液转移到旋转柱上, 用1000µL 的吸管和离心机在 6000 x g 处, 1 分钟25摄氏度。</li>
	<li>将旋转柱放在一个干净的2毫升收集管与戴手套的手, 并丢弃的收集管包含的流动进入一个塑料处理箱。</li>
	<li>用1000µL 的吸管和离心机将500µL 的洗涤缓冲器添加到旋转柱上, 在25摄氏度时, 在 6000 x g 处为1分钟。</li>
	<li>将旋转柱放在一个干净的2毫升收集管与戴手套的手, 并丢弃的收集管包含的流动进入一个塑料处理箱。</li>
	<li>用1000µL 的吸管和离心机将500µL 的洗涤缓冲器添加到旋转柱上, 在25摄氏度时, 在 6000 x g 处为1分钟。</li>
	<li>将包含流经的收集管丢弃到塑料处理箱中。将旋转柱放在干净的1.5 毫升离心管中, 用手套的手和离心机在 2万 x g 3 分钟在25°c, 以干燥膜。</li>
	<li>将旋转柱放置在 DNA 低结合管中, 戴手套的手。<ol>
<li>添加 40-50 µL 洗脱缓冲液的中心膜与100µL 吸管。</li>
		<li>在室温下孵育5分钟, 离心机在 2万 x g 处为1分钟, 25 摄氏度。</li>
		<li>将 DNA 样本存储在-20 摄氏度, 直到下一步 (该协议可以在此暂停)。</li>
	</ol>
</li>
</ol>4. 定量实时 PCR 法估计 DNA 质量
<ol><li>用吸管制备无菌离心管中的主混合, 如下所示:10 µL 2x 实时 PCR 总混合, 1 µL 20x RNase P 底漆-探针混合 (扩增子尺寸:87 bp), 8 µL 无菌核酸酶水。</li>
	<li>在一个无菌离心管中准备第二个主组合, 如下所示:10 µL 的2x 实时 PCR 主混合, 1 µL 20x RNase P 底漆-探针混合 (扩增子大小: 268 bp), 8 µL 无菌核酸酶水。</li>
	<li>执行人类控制基因组 DNA 的串行稀释 (在试剂盒中提供) 4 次, 用于五点标准曲线并确定绝对 DNA 浓度13。</li>
	<li>将两个准备好的主混合 (在步骤4.1 和4.2 中准备) 的19µL 添加到具有20µL 吸管的光学96阱反应板的分离井中。</li>
	<li>添加1µL 的 FFPE dna 或 TIC dna, 以分离含有与 2-µL 吸管的反应混合物的水井。将1µL 的无核酸酶水添加到一个单独的井中, 其中包含没有模板控制的反应组合。</li>
	<li>保持光学胶膜的非粘接面, 并从胶片的中心剥离保护背衬。轻轻地将涂抹器拖到胶片上, 并将胶片密封在96井板上。</li>
	<li>在十年代的室温下, 以2000转每分钟的温度, 用96井板式搅拌机轻轻地混合96井板。在室温下, 离心板以 1000 x g 为3分钟。</li>
	<li>对实时 PCR 仪进行电源, 并插入96井板。使用以下协议运行 PCR 反应:95 °c 二十年代, 其次45周期95°c 为 1 s 和60°c 为二十年代. 使用 "标准曲线" 和 "快速模式"。</li>
	<li>用 dna 比值 (相对量化) 评估 dna 碎片;RQ) 获得的长扩增子 (268 bp) 到短扩增子 (87 bp)。RQ 是长扩增子的平均值/短扩增子13的平均值。</li>
</ol>5. 下一代测序库的编制
<ol><li>根据制造商的说明, 为下一代测序准备序列库。</li>
	<li>在无菌离心管中制备多路 pcr 主混合体, 如下所示: 4 µL 的5x 多重 pcr 反应溶液, 4 µL 5x 引物池, ≤6µL TIC 或 FFPE DNA (1-100 ng), 并添加核酸酶免费水多达20µL。<ol>
<li>将多路 pcr 主混合添加到 pcr 管中, 通过轻敲管轻轻混合。</li>
		<li>在室温下用微型离心机将离心管在 1500 x g 处旋转5秒。</li>
	</ol>
</li>
	<li>使用以下协议运行 PCR 反应:99 °c 为2分钟, 其次20个周期99°c 为十五年代和60°c 为4分钟和保持步骤10°c。在室温下, 用 1500 x g 的微型离心机将 PCR 管简单地旋转到5秒。 注: 根据底漆对的数量确定循环次数。</li>
	<li>打开 PCR 管的盖子, 并添加2µL 的限制酶与2µL 的吸管。闭上 pcr 管的盖子, 轻轻地用 pcr 管轻拌。在室温下, 用 1500 x g 的微型离心机将 PCR 管简单地旋转到5秒。</li>
	<li>使用以下协议运行 PCR 反应:50 °c 为10分钟, 55 °c 为10分钟, 60 °c 为20分钟, 并且保持步骤10°c。在室温下, 用 1500 x g 的微型离心机将 PCR 管简单地旋转到5秒。</li>
	<li>将适配器结扎主组合添加到包含已消化 PCR amplicons 的每个井中, 如下所示: 4 µL 适配器结扎液, 0.5 µL 条码, 0.5 µL 适配器, 2 µL 无核酸酶水, 2 µL DNA 连接酶。关闭 PCR 管的盖子, 轻轻地搅拌。在室温下, 用 1500 x g 的微型离心机将 PCR 管简单地旋转到5秒。</li>
	<li>使用以下协议运行 PCR 反应:22 °c 为30分钟, 68 °c 为5分钟, 72 °c 为5分钟, 并且保持步骤10°c。</li>
	<li>根据制造商的指示, 用磁性珠子净化测序库。</li>
	<li>将适配器结扎的库解决方案转移到1.5 毫升的 DNA 低结合管中。在 1st纯化的 DNA 低结合管中添加45µL 磁性珠子。轻轻地搅拌, 在室温下孵化5分钟。</li>
	<li>将 DNA 低结合管放置在磁性机架中, 然后在室温下孵化2分钟, 直到溶液清晰为止。在不干扰磁性珠子的情况下, 用200µL 吸管小心地丢弃上清液。</li>
	<li>添加150µL 新鲜准备的70% 乙醇与一个200µL 的吸管, 然后移动管侧的磁铁, 以洗涤珠。小心丢弃上清, 不干扰磁性珠子。</li>
	<li>重复步骤5.11 以进行第二次清洗。</li>
	<li>在室温下, 用迷你离心机在 1500 x g 处向下旋转管, 5 秒。将 DNA 低结合管放置在磁性机架中, 用10µL 吸管小心地丢弃乙醇液滴。</li>
	<li>将50µL 的低 TE 加入到含有磁性珠子颗粒的 DNA 低结合管中, 以驱散珠子。室温下孵育2分钟。</li>
	<li>将 DNA 低结合管放置在磁性机架中, 在室温下孵育2分钟, 直到溶液清晰为止。</li>
	<li>将50µL 的上清液转移到新的 DNA 低结合管中, 并在 2nd纯化中添加75µL 的磁性珠子和100µL 吸管。轻轻地搅拌, 在室温下孵化5分钟。</li>
	<li>将 DNA 低结合管放置在磁性机架中, 然后在室温下孵育2分钟, 直到溶液清晰为止。在不干扰磁性珠子的情况下, 用200µL 吸管小心地丢弃上清液。</li>
	<li>添加150µL 新鲜准备的70% 乙醇与一个200µL 的吸管, 然后移动管侧的磁铁, 以洗涤珠。小心丢弃上清, 不干扰磁性珠子。</li>
	<li>重复步骤5.18 以进行第二次清洗。</li>
	<li>在室温下, 用微型离心机在 1500 x g 处以5秒的温度旋转管子。将 DNA 低结合管放置在磁性机架中, 用10µL 吸管小心地丢弃乙醇液滴。</li>
	<li>将50µL 的低 TE 加入到含有磁性珠子颗粒的 DNA 低结合管中, 以驱散珠子。室温下孵育2分钟。</li>
	<li>将 DNA 低结合管放置在磁性机架中, 在室温下孵育2分钟, 直到溶液清晰为止。</li>
	<li>将含有纯化库的45µL 的清液转移到新的 DNA 低结合管中, 用100µL 的吸管。</li>
</ol>6. 定量实时 PCR 量化图书馆集中度
<ol><li>根据制造商的说明13确定每个库的集中程度。<ol>
<li>准备一个20倍稀释溶液如下: 混合2µL 纯化的图书馆和38µL 核酸酶无水的 DNA 低结合管与2µL 和100µL 吸管。</li>
		<li>将未稀释的库存储在-20 °c, 直到步骤7.3。</li>
	</ol>
</li>
	<li>准备一个200倍稀释溶液如下: 混合5µL 的20倍稀释纯化库 (准备在步骤 6.1) 和45µL 核酸酶无水的 DNA 低结合管。</li>
	<li>准备一个2000倍稀释溶液如下: 混合5µL 的200倍稀释纯化库 (准备在步骤 6.2) 和45µL 核酸酶无水的 DNA 低结合管。</li>
	<li>准备反应大师组合如下: 混合10µL 2x 主混合溶液和1µL 20x 底漆探针检测溶液在无菌离心管与吸管, 然后混合通过攻丝管。将反应主混合的11µL 加入光学96阱反应的井中。</li>
	<li>添加9µL 2000 倍稀释库, 每标准控制9µL, 或9µL 的核酸酶水, 每井与10µL 吸管。</li>
	<li>保持光学胶膜的不粘接面, 并从胶片的中心剥离保护背衬。<ol>
<li>轻轻地将涂抹器拖到胶片上, 并将胶片密封在96井板上。</li>
		<li>在室温下, 在十年代用96井板式搅拌机轻轻混合96井板。</li>
		<li>在室温下, 离心板以 1000 x g 为3分钟。</li>
	</ol>
</li>
	<li>对实时 PCR 仪进行电源, 并插入96井板。使用以下协议运行 PCR 反应:50 °c 为2分钟, 95 °c 为二十年代, 跟随40个周期95°c 为 1 s 和60°c 为二十年代. 使用 "标准曲线" 和 "快速模式"。</li>
	<li>通过将 qPCR 确定的浓度乘以 2000, 计算未稀释的库浓度。</li>
</ol>7. 下一代测序
<ol><li>计划运行条件并在软件中设置运行参数。<ol>
<li>单击 [计划选项卡] 和 [模板], 然后选择适当的运行方法。</li>
		<li>选择应用程序和技术类型, 然后单击 [下一步]。</li>
		<li>选择仪器、样品准备套件 (可选)、库套件类型、模板套件、排序工具包、基本校准模式、芯片类型、控制序列 (可选) 和条形码集, 然后单击 [下一步]。</li>
		<li>选择插件, 然后单击 [下一步]。</li>
		<li>选择项目并单击 [下一步]。</li>
		<li>选择目标区域的默认参考和床文件。</li>
		<li>键入示例名称, 选择条形码, 然后单击 [计划运行]。</li>
	</ol>
</li>
	<li>根据制造商的指示, 在自动仪器中执行模板准备和芯片加载。使用前将试剂盒在室温下解冻45分钟。</li>
	<li>根据步骤6.8 中计算的库集中度和晚上20点的图书馆, 稀释未稀释的图书馆与无核酸酶的水。<ol>
<li>准备一个汇集库, 用于排序和储存在冰上。</li>
		<li>增加25µL 的汇集图书馆与100µL 吸管在样品管的底部。在48小时内使用池库。</li>
	</ol>
</li>
	<li>开启并打开自动仪器的盖子。<ol>
<li>将测序芯片、芯片适配器、浓缩盒、尖端墨盒、pcr 板、pcr 框架密封、回收管、溶液盒和试剂盒放到自动仪器的适当位置。</li>
		<li>在屏幕上触摸 [设置运行] 和 [逐步]。</li>
		<li>在屏幕上关闭盖子并触摸 [开始检查]。</li>
		<li>在甲板扫描过程之后, 触摸屏幕上的 [下一页]。</li>
		<li>检查显示内容 (套件类型、芯片类型、芯片 id、样本 id、计划)、设置时间和触摸屏幕上的 [确定]。</li>
	</ol>
</li>
	<li>完成芯片加载后:<ol>
<li>触摸 [下一步] 在屏幕上打开盖子。</li>
		<li>从芯片适配器卸下带手套的手的测序芯片。</li>
		<li>将芯片放入芯片容器, 说唱与 parafilm 和存储在4°c, 直到排序反应。</li>
		<li>从自动仪器的适当位置取出浓缩盒、pcr 板、pcr 框架密封、回收管、溶液盒和试剂盒, 戴手套的手。</li>
		<li>将空尖端墨盒转移到自动仪器的废料尖端位置, 戴手套的手。</li>
		<li>触摸 [下一步] 并关闭盖子。</li>
		<li>触摸 [开始] 和清洁自动仪器的紫外线4分钟。</li>
	</ol>
</li>
	<li>用0.22 µm 滤池过滤器将亚氯酸钠片溶解在1000毫升的超纯水和过滤液中。<ol>
<li>在测序仪上通电。</li>
		<li>在测序仪的屏幕上触摸 [清洁] 和 [下]。</li>
		<li>用250毫升过滤灭菌的亚氯酸钠溶液和随后250毫升的超纯水清洗测序仪。</li>
	</ol>
</li>
	<li>触摸 [初始化] 并在屏幕上选择适当的排序工具包。<ol>
<li>安装一个灰色托运人在适当的位置戴手套的手。</li>
		<li>用洗涤液 (套件中提供)、ph 调节溶液 (含350µL 100 毫米氢氧化钠) 和 ph 标准溶液 (在试剂盒中提供) 初始化测序仪。</li>
	</ol>
</li>
	<li>在50毫升的试管中添加20µL 的 dATP、dGTP、dCTP 和 dTTP 核苷酸 (在套件中提供), 用 100-µL 吸管。<ol>
<li>安装一个灰色托运人在适当的位置戴手套的手。</li>
		<li>将50毫升管和螺钉装入测序仪。</li>
		<li>触摸屏幕上的 [下一页] 开始初始化步骤, 大约需要25分钟。</li>
	</ol>
</li>
	<li>完成初始化步骤后:<ol>
<li>在屏幕上触摸 [运行] 并选择适当的库准备工具。</li>
		<li>扫描芯片的二维条码。</li>
		<li>在适当的位置插入测序芯片。</li>
		<li>关闭芯片钳和仪表门。</li>
		<li>触摸 [芯片检查], [下一页] 和 [OK] 在屏幕上开始排序运行。</li>
	</ol>
</li>
	<li>在排序反应之后, 在排序服务器13、17上传输数据并执行数据分析管道。</li>
</ol>

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作者没有什么可透露的。

在本研究中, 我们提出了一种从临床病理标本中获取肿瘤 DNA 的替代方法。TIC 准备是非常简单的, 需要较少的时间与 FFPE 方法相比, 没有特殊仪器的要求10。从 TIC 准备到 DNA 提取的所有程序都可以在两天内完成 (图 1)。因此, 这种方法缩短了进行基因检测的周转时间。值得注意的是, 这为缩短分子分析所需的天数提供了重要的优势。这一短暂的周转时间使我们?...

下一代测序技术为研究人员在基因变异、孟德尔病、遗传性倾向和癌症方面的基因组信息分析提供了重要的进展1,2,3.癌症基因图谱 (TCGA) 和国际癌症基因组联合会 (ICGC) 一直在寻找几种常见癌症的基因改变的鉴定4。数以百计的基本癌症驱动基因已被成功地确定, 其中一些分子被瞄准药物开发1,5,6。
在临床环境中, FFPE 标本通常用于各种疾病, 包括癌症的病理诊断和分子检测。然而, 在福尔马林固定过程中, dna 蛋白或 dna dna 交联发生, 导致 dna 分裂。因此, FFPE dna 样本并不总是适合遗传分析, 因为质量和数量的 dna7,8,9。此外, 准备 FFPE 标本需要数天的时间, 技术技能是准确准备剖面的必要条件。因此, 建立一种简便、快速的获取高质量完整 DNA 的方法是可取的。
细胞学是病理诊断的一种替代方法。与 FFPE 准备10相比, 细胞学样品制备方法更简单、成本更低、更快速。采用 TIC 技术对乳腺癌患者的前哨淋巴结和边缘组织进行了一些年的术中快速诊断, 其方法为11,12。然而, 很少有报告研究了高质量的基因组 DNA 是否可以从 TIC 标本中提取, 并用于后续的遗传分析。细胞学标本通常染色阴道 (Pap) 或姬姆萨染色, 我们以前报告说, 从 TIC 标本 (特别是姬姆萨染色样品) 提取的 DNA 的数量和质量优于从 FFPE 获得的样本组织13。与 Pap 染色相比, 姬姆萨染色在要求较少染色的过程中具有优势。在 Pap 染色, 样品被固定和染色后, 必须安装在安装培养基 (例如, Malinol), 以区分样本内容, 如肿瘤细胞, 正常细胞和炎症细胞在显微镜下。如果在没有安装步骤的情况下制备 Pap 标本, 在显微镜下观察细胞几乎是不可能的, 因为试样是干的。相比之下, 姬姆萨染色可以观察到干燥状态, 因此, 安装步骤是不必要的快速细胞评估。对于显微切割, 姬姆萨染色更适合, 因为它需要干燥标本。
在本报告中, 我们介绍了一个简单快速的方法来制备 tic 标本与姬姆萨染色, 并表明 TIC 是一个更好的来源的 DNA 比较 FFPE 标本。

<table><tbody><tr><td>FINE FROST white20 micro slide glass</td><td>Matsunami Glass ind, Ltd</td><td>SFF-011</td><td></td></tr><tr><td>Arcturus PEN Membrane Glass Slides</td><td>Thermo Fisher Scientific</td><td>LCM0522</td><td></td></tr><tr><td>Cyto Quick A solution</td><td>Muto Pure Chemicals</td><td>20571</td><td></td></tr><tr><td>Cyto Quick B solution</td><td>Muto Pure Chemicals</td><td>20581</td><td></td></tr><tr><td>May-Grunwald Solution</td><td>Muto Pure Chemicals</td><td>15053</td><td></td></tr><tr><td>Giemsa solution</td><td>Muto Pure Chemicals</td><td>15002</td><td></td></tr><tr><td>QIAamp DNA FFPE tissue kit</td><td>Qiagen</td><td>56404</td><td></td></tr><tr><td>TaqMan Fast Advanced Master Mix</td><td>Thermo Fisher Scientific</td><td>4444557</td><td></td></tr><tr><td>TaqMan RNase P Detection Reagents Kit</td><td>Thermo Fisher Scientific</td><td>4316831</td><td></td></tr><tr><td>TaqMan Assay from FFPE DNA QC Assay v2</td><td>Thermo Fisher Scientific</td><td>4324034</td><td></td></tr><tr><td>MicroAmp Fast Optical 96-Well Reaction Plate&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>4346907</td><td></td></tr><tr><td>MicroAmp optical Adhesive Film</td><td>Thermo Fisher Scientific</td><td>4311971</td><td></td></tr><tr><td>MicroMixer E36</td><td>TITEC</td><td>0027765-000</td><td></td></tr><tr><td>ViiA 7 Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>VIIA7-03</td><td></td></tr><tr><td>Himac CF16RXII</td><td>Hitachi-koki</td><td>CF16RII</td><td></td></tr><tr><td>Ion Library TaqMan Quantitation Kit</td><td>Thermo Fisher Scientific</td><td>4468802</td><td></td></tr><tr><td>Ion AmpliSeq Cancer Hotspot Panel v2</td><td>Thermo Fisher Scientific</td><td>4475346</td><td></td></tr><tr><td>Ion AmpliSeq Library Kit 2.0</td><td>Thermo Fisher Scientific</td><td>4480442</td><td></td></tr><tr><td>Ion Xpress Barcode Adapters 1-16 Kit</td><td>Thermo Fisher Scientific</td><td>4471250</td><td></td></tr><tr><td>Ion PGM Hi-Q View Sequencing Kit (200 base)</td><td>Thermo Fisher Scientific</td><td>A30044</td><td></td></tr><tr><td>Ion Chef System</td><td>Thermo Fisher Scientific</td><td>4484177</td><td></td></tr><tr><td>Veriti 96-well Thermal Cycler</td><td>Thermo Fisher Scientific</td><td>Veriti200</td><td></td></tr><tr><td>Ion 318 Chip Kit v2 BC</td><td>Thermo Fisher Scientific</td><td>4488150</td><td></td></tr><tr><td>Ion PGM System</td><td>Thermo Fisher Scientific</td><td>PGM11-001</td><td></td></tr><tr><td>Ion PGM Wash 2 Bottle kit</td><td>Thermo Fisher Scientific</td><td>A25591</td><td></td></tr><tr><td>Agencourt&amp;trade; AMPure&amp;trade; XP Kit</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>16-position Magnetic Stand</td><td>Thermo Fisher Scientific</td><td>4457858</td><td></td></tr><tr><td>Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)</td><td>Thermo Fisher Scientific</td><td>AM12450</td><td></td></tr><tr><td>Nuclease-free water</td><td>Thermo Fisher Scientific</td><td>AM9938</td><td></td></tr><tr><td>MicroAmp&amp;trade; Optical 96-well Reaction Plates</td><td>Thermo Fisher Scientific</td><td>4306737</td><td></td></tr><tr><td>MicroAmp&amp;trade; Clear Adhesive Film</td><td>Thermo Fisher Scientific</td><td>4306311</td><td></td></tr><tr><td>Agencourt&amp;trade; AMPure&amp;trade; XP Kit</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Ethanol(99.5)</td><td>Nacalai Tesque</td><td>08948-25</td><td></td></tr><tr><td>Sodium hydroxide (10M)</td><td>Sigma</td><td>72068</td><td></td></tr><tr><td>DTU-Neo</td><td>TAITEC</td><td>0063286-000</td><td></td></tr><tr><td>E-36</td><td>TAITEC</td><td>0027765-000</td><td></td></tr><tr><td>ECLIPSE Ci-L</td><td>Nikon</td><td>704354</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-2XLS+</td><td>METTLER TOLEDO</td><td>17014393</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-10XLS+</td><td>METTLER TOLEDO</td><td>17014388</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-20XLS+</td><td>METTLER TOLEDO</td><td>17014392</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-100XLS+</td><td>METTLER TOLEDO</td><td>17014384</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-200XLS+</td><td>METTLER TOLEDO</td><td>17014391</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-1000XLS+</td><td>METTLER TOLEDO</td><td>17014382</td><td></td></tr><tr><td>petit-change</td><td>WAKEN</td><td>MODEL8864</td><td>Mini centrifuge</td></tr><tr><td>petit-incubator</td><td>WAKEN</td><td>WKN-2290</td><td>Air incubator</td></tr><tr><td>SensiCare Powder-free Nitrile Exam Gloves</td><td>MEDLINE</td><td>SEM486802</td><td></td></tr><tr><td>Sterile gauze</td><td>Osaki</td><td>11138</td><td></td></tr><tr><td>Refrigerator MediCool</td><td>SANYO</td><td>MPR-312DCN-PJ</td><td></td></tr><tr><td>FEATHER TRIMMING BLAD</td><td>FEATHER</td><td>No.130</td><td></td></tr><tr><td>FEATHER TRIMMING BLAD</td><td>FEATHER</td><td>No.260</td><td></td></tr><tr><td>FEATHER S</td><td>FEATHER</td><td>FA-10</td><td></td></tr><tr><td>Vortex Genius 3</td><td>IKA</td><td>41-0458</td><td>Vortex mixer</td></tr><tr><td>Pincette</td><td>NATSUME</td><td>A-5</td><td></td></tr><tr><td>1.5 mL microtube</td><td>BIOBIK</td><td>RC-0150</td><td></td></tr></tbody></table>

simple and rapid method to obtain high-quality tumor dna from clinical-pathological specimens using touch imprint cytology

在癌症患者的特定分子靶向药物的治疗前, 确定肿瘤的突变状态是至关重要的。在临床环境中, 福尔马林固定石蜡 (FFPE) 组织广泛用于基因检测。然而, FFPE DNA 在福尔马林固定过程中通常会受到损伤和分裂。因此, 由于 dna 质量和数量的降低, FFPE dna 有时不足以进行基因检测。在这里, 我们提出了触摸印记细胞学 (TIC) 的方法, 以获得肿瘤细胞的基因组 DNA, 可以在显微镜下观察到。细胞形态学和癌细胞数量可以用 TIC 标本来评估。此外, 从 TIC 样品中提取基因组 DNA 可以在两天内完成。利用该方法获得的 TIC dna 的总量和质量均高于 FFPE dna。这种快速简便的方法使研究人员能够获得高质量的基因检测 DNA (例如、下一代测序分析、数字 pcr 和定量实时 pcr), 并缩短报告结果的周转时间。

图 1显示了从准备 TIC 标本到 DNA 提取的整个过程。值得注意的是, 该手术仅需两天时间才能从 TIC 样品中获得基因组 DNA。我们评估了在滑动处理前肿瘤贮存的任何影响。我们发现, 当组织标本立即触及到幻灯片上时, 肿瘤细胞附着在玻璃滑梯上, 当组织被保存在生理盐水湿润的无菌纱布中时, 1 小时 (图 2)。然而, 当组织保持在室温...

Watch this Scientific Journal Video about Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology at JoVE.com

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

从肿瘤组织获得高质量的基因组 DNA 是利用下一代测序分析基因改变的重要第一步。本文提出一种简便、快速的方法来丰富肿瘤细胞, 从接触印记细胞学标本中获得完整的 DNA。

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. ...

simple-rapid-method-to-obtain-high-quality-tumor-dna-from-clinical

Research

JoVE Journal

Cancer Research

10.8K 次观看.  Yamanashi Central Hospital. 从肿瘤组织获得高质量的基因组 DNA 是利用下一代测序分析基因改变的重要第一步。本文提出一种简便、快速的方法来丰富肿瘤细胞, 从接触印记细胞学标本中获得完整的 DNA。

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

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

新一代测序为可操作的突变在固体和液体瘤检测

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

科研

癌症研究

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

从病人的血液样本可行的循环肿瘤细胞的快速分离

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

生物工程

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

通过有针对性的测序方法进行比较病变分析

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

行为学

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

免疫与感染

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

生物学

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic &#8216;Touch DNA&#8217; Evidence

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a &#8216;blind-swabbing&#8217; approach will co-sample cellular material from the different individuals, even if the individuals&#8217; cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim&#8217;s DNA may be found in significant excess thus masking any potential perpetrator&#8217;s DNA.
In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, &#8216;smart analysis&#8217; method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 &#181;l microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., &#8220;clumps&#8221;) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Here we describe an optimized and efficient removal strategy for the collection of bio-particles present in &#8216;touch DNA&#8217; samples, together with an enhanced amplification protocol involving a one-step 5 &#181;l micro-volume lysis/STR amplification, to permit the recovery of short tandem repeat (STR) profiles of the bio-particle donor(s).

单人的生物粒子从法医"触摸DNA&#39;证据回收简体显微增强遗传分析

DNA profiles can be obtained from &#8216;touch DNA&#8217; evidence, which comprises microscopic traces of human biological material. Current methods for ...

Enhancing Tumor Content through Tumor Macrodissection

The presence of contaminating non-tumor tissues in formalin-fixed paraffin-embedded (FFPE) tissues can greatly undermine genomic studies. Herein we describe macrodissection, a method designed to augment the percentage tumor content of a tissue specimen by removing and eliminating unwanted tissue prior to performing downstream nucleic acid extractions. FFPE tissue blocks were sectioned to produce 4-5 &#181;m slide-mounted tissue sections. A representative section was submitted for hematoxylin and eosin (H&#38;E) staining and subsequently reviewed by a board-certified pathologist. During the review, the pathologist identified and marked the regions of tumor tissue in the H&#38;E. Once complete, the demarked H&#38;E was used to guide resection of the serial unstained sections from the same tissue block. To demonstrate the effects of macrodissection, RNA extracted from matched macrodissected and non-dissected Diffuse Large B-Cell Lymphomas (DLBCL) were run on a digital gene expression assay capable of determining DLBCL subtype and BCL2 translocation status. The results showed that macrodissection changed the subtype or BCL2 translocation status calls in 60% of the samples examined. In conclusion, macrodissection is a simple and effective method for performing tumor enrichment prior to nucleic acid extractions, the product of which can then be confidently used in downstream genomic studies.

This protocol presents a method to increase the percent tumor content of formalin-fixed paraffin-embedded tissue samples.

通过肿瘤大解剖增强肿瘤含量

The presence of contaminating non-tumor tissues in formalin-fixed paraffin-embedded (FFPE) tissues can greatly undermine genomic studies. Herein we ...