这项研究得到荷兰癌症协会 (阿尔卑斯 2013-6371) 和 VONK Egbers 基金会的支持。我们感谢荷兰反洗钱组织工作组的协作和卓有成效的讨论, 以持续改进和标准化反洗钱。

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屋宇署亲切地提供了若干独立研究所用于证实干细胞管的抗体鸡尾酒。作者对这篇手稿没有什么可透露的。

有充足的数据可以证明 mrd 阳性与生存不良有关, 因此 mrd 评估可以通过提供额外的预后信息来提高患者预后, 从而可以做出临床决定。因此, 对 MRD 进行免疫表型评估的一致统一方法对于最终改善患者治疗至关重要。在比较不同临床部位的临床研究时, 这一点也很重要, 最终可能有助于临床决策, 并作为总生存的替代临床终点。有必要为精确和一致的 MRD 测量提供坚实的指导方针, 这一概念导致欧洲白血病网络 (民族解放军) 协同行动, 设计了艺术指南的现状。这份全面的文件将 late-2017 出版, 将有助于许多研究小组和实验室向前迈进。
议定书内的关键步骤
mrd 分析的一个重要而经常被忽视的方面是样品质量对 mrd 准确测定的影响。鉴于在这一背景下需要考虑到额外的操作考虑, 在全球临床试验中必须将材料运送到其他研究所, 这一点更明显。为了减少混淆患者信息和及时提供样本的风险, 适当的管理是至关重要的。再次, 在运输的这个阶段, 正确的形式和/或导入到电子医院系统中, 要求分析的明确任务是必需的。注意到对 MRD 取样治疗的阶段也至关重要, 因为它可能与临床决策有关 (例如在第二疗程后), 因此应明确界定。模拟数据的使用 (例如, 出生年的 1月1日) 增加了混合病人或分析的风险。如法律要求, 应采用另一种匿名的身份查验方式。
所有免疫分型的标本应优先处理24小时内收集。虽然不能推荐, 但在常温下, 骨髓和外周血标本仍可进行处理和分析。此外, 所有搬运的材料都可以在无菌条件下进行, 因此 (局部) 生物库细胞的进一步超低温保存仍然是相关的: 许多临床研究还有进一步的分析, 以进一步研究白血病细胞与关于分子、免疫表型和功能性功能, 将在稍后阶段进行。然而, biobanking 的细节不在本手稿的范围之内。
使用 mrd 作为诊断工具意味着它需要一个认可的实验室, 不仅用于流式细胞术, 而且还涉及对 MRD 评估的质量控制。这可能需要向特定的参考实验室分发样本, 或者 (重新) 通过参考小组的 MRD 测量来分析流程文件。
本文介绍了从骨髓穿刺收集到测定临床样品中的 MRD 的最重要的行动-需要一整个专家小组的具体任务, 责任, 并经常沟通, 以有效表征和分析。由于每项任务对程序都至关重要, 因此建议在每个实验室都有健全的后勤, 包括各项议定书, 以及为这项任务专门培训的足够的备份人员。此外, 由于部分程序中有一些相对主观的步骤 (特别是 LAIP 和的异常标记识别), 因此必须对团队的最终结果进行讨论, 并有一个小组授权的最终报告。主管.目前的努力正在寻求计算机软件开发, 这将有助于标准化 (LAIP 和) MRD 分析。
修改和疑难解答
每个实验室都可以有它自己的一套抗体, 可以用来定义不同的亚人口, 虽然有一个标准化的标志骨干是必不可少的可比结果, 如民族解放军的艺术准则的《 MRD 测量文件》概述上文所述。无论选择的标记有哪些问题, 都可以使分析结果变得困难, 需要加以考虑。
技术的局限性
在诊断和监测期间 (在治疗期间和之后), 首先评估 LAIP 和基因检测异常标记表达的鉴别, 以准确描述 MRD 表型。虽然超过95% 的患者可以通过 LAIP 或 (或两者) 评估, 但仍有一些患者没有定义的 LAIPs 或没有 CD34+CD38 肝干细胞或存在 CD34 阴性爆炸, 或有一个失踪的诊断样本。 在这些情况下, 仍然值得尝试和测量 MRD 与一个抗体的面板广泛, 因为可用细胞的数量允许, 然后选择最可靠 (给予白血病细胞最强的区别) LAIP。考虑到最终复发患者的比例并不完全类似于诊断免疫表型, 由于 AML 疾病的异质性, 因此建议在 MRD 测量一个广泛的抗体组。这些免疫表型转移包括爆炸细胞和肝干细胞, 并已被证明发生在治疗16和重大疾病可能基于这些所谓的即将到来的人群。然而, 目前还没有确定所有免疫表型的亚人口是否会导致疾病复发, 因此不常见的做法是报告根据这些细胞在临床试验中的 MRD 定制疗法。重要的是要注意到, 由于人口转移而导致的缺音的风险减少了, 其中最重要的偏航定义标记在一个流式细胞仪 (PE) 通道14中的一个管方法。
关于现有方法的意义
本协议中描述的方法依赖于一个或多个 LAIPs 的定义, 在治疗过程中遵循细胞。这种方法的缺点是最近已经表明 LAIP 可能会在治疗过程中发生变化。这样, 一些有不同异常标记的即将到来的人群可能会被遗漏。为了规避这种情况, 最好是测量所有异常标记, 而不是仅检测诊断结果。这将类似于几个实验室17使用的 "不同于正常" 方法。
未来应用
近年来, 在新的临床试验设计中, MRD 受到了格外的关注。在专门治疗方案和靶向药物治疗的时代, 使用 MRD 作为结果措施将减少建立新治疗的临床疗效所需的时间, 最终允许更快地引入急需的治疗方法。选择进入诊所。适当的后勤和实际执行统一的 MRD 评估的意义对今后的反洗钱治疗成功至关重要, 因为 FDA 目前正在调查使用 MRD 作为替代终点的可行性, 而不是全面生存。度量值18。

急性髓系白血病 (AML) 是一种恶性肿瘤的骨髓的特点是缺陷的成熟程序, 与异常增殖和积累的髓祖细胞, 抑制正常造血, 最终骨髓衰竭.这种疾病在形态学、免疫表型、细胞遗传学、分子畸变、基因表达特征以及治疗反应和治疗结果方面高度异构,1,2。目前的管理包括诱导化疗, 目的是实现完全缓解 (CR), 其次是缓解后治疗, 这主要是由分子, 细胞遗传学和免疫表型研究的结果, 包括任何若干疗程的额外化疗或 (自体或同种异体) 干细胞移植3。 尽管在90% 的密集化疗后的高缓解率, 成人5年存活率仅约 30%-40%, 主要是由于复发的发展, 通常是耐化疗, 从而很难治疗。结果在孩子是更好的, 虽然大约一第三也复发。因此, 及早发现即将复发将填补未满足的医疗需求, 并可能指导缓解后治疗4。
治疗后残留疾病可能反映所有诊断和诊断后耐药性机制/因素的总和;因此, 它的测量可能是预后的, 有助于指导治疗。deﬁning 残留疾病的可能性 (以前称为最小残留疾病, 现在称为可测量残留疾病或 MRD) 远远低于5% 个爆炸细胞的形态学标准是改变风险 classiﬁcation 的景观。目前主要用于检测 MRD 的两种方法是基于流式细胞仪和分子基础的, 后者由逆转录酶 PCR (RT qPCR)5评估, 或者, 尽管在过早阶段, 由下一代测序 (。许多成人和儿童的研究已经表明, 在归纳和整合治疗后, 各种 MRD 方法在反洗钱中提供了强有力的预后信息6,7, 以及疾病负担的新定义 (优于形态学 CR) 现在正在出现8。这表明, 通过流动和/或分子技术评估的 MRD 应该成为, 事实上已经成为标准的每一个临床试验中的 AML。
本文讨论了详细的流式细胞术程序, 以获得准确和可重复的免疫表型特征的骨髓标本, 包括骨髓取样和处理程序前流式细胞术。高质量的骨髓样本在诊断和随访中的可用性对于这项测量在临床和临床试验中的成功至关重要。事实上, 这些前期分析的考虑对于分子 (PCR 和) MRD 的方法也是至关重要的。对于 MRD 的免疫表型特征, aberrantly 表达标记与正常髓样和祖标记结合, 以确定白血病相关的免疫表型 (LAIP)9。MRD 测量影响治疗反应的许多因素的结果, 如内在或获得的白血病耐药性, 治疗的药效学和动力学, 免疫监视和依从性。因此, MRD 是一个很强的后诊断预后参数相关的临床结果时, dichotomized 的截止水平, 由接受手术的特点 (ROC) 分析。对于我们的成人 AML 队列的 HOVON 42a 研究, 切断水平是设置在0.1% 的 LAIP 阳性细胞/总白细胞。使用这个标准来确定阴性与阳性的 MRD 状态, 一组患者可以确定谁有明显更严重的复发率, 无复发和整体生存6。此外, 我们描述的测量不成熟, 耐药白血病细胞的干细胞样特征 (CD34+CD38 白血病干细胞, 或), 它提供了一个强有力的预测患者的结果10。LAIP 和细胞方法一起形成了流程。LAIP 方法适用于大约90% 的患者, 而在约80% 的患者中, 可采用方法。95% 以上的病人可以为一个参数或两者一起评估。
最后, 本刊物提供了一个详细的操作描述, 以评估 MRD 的流式细胞术。这包括: 1) 骨髓取样程序的统一和/或标准化, 2) 样品运输指南 3) 使用多种抗体板 (包括单细胞管法) 对白血病细胞检测的详细描述4) 为标准测量、5) 对 MRD 测量的分析程序和 6) 对检测的分析程序建立了对其进行标准化测试的流化机的特征。
我们的目的是显示过程的所有方面, 包括样品准备, 因为这是很少讨论, 而它是一个重要的问题, 最终结果的质量。骨髓穿刺和活检是用于评估骨髓造血细胞的临床程序。这些与完整的血液计数 (CBC) 和血液涂片一起执行。骨髓穿刺的最佳方法是准确诊断和随访对 MRD 测量的关键。此外, 一个成功的骨髓穿刺应该包含足够的细胞来执行 LAIP 和外流分析 (至少1000万活细胞)。在这里, 我们描述了执行骨髓穿刺的方法, 并提供了指导, 这应该导致足够的细胞取样 (并限制血液稀释的可能性), 以进行准确的诊断和额外的研究。这些前期分析的考虑对于分子 (PCR 和) MRD 的方法也是至关重要的。所有免疫分型的标本应优先处理24小时内收集。虽然不能推荐, 但在常温下, 骨髓和外周血标本仍可进行处理和分析。此外, 所有搬运与材料应在无菌条件下进行, 以使不育细胞的低温保存, 以供日后研究/质量评估等。

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			<td ></td>
			<td>BD Biosciences</td>
			<td>335039</td>
			<td>CD45RA FITC</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >345810</td>
			<td >CD56 PE</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >337899</td>
			<td >CD22 PE</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >345785</td>
			<td >CD14 PE</td>
		</tr>
		<tr >
			<td ></td>
			<td >Miltenyi Biotec</td>
			<td >130-080-801</td>
			<td >CD133 PE</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td>562566</td>
			<td>Clec12a PE</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td>565568</td>
			<td>Tim-3 PE</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >332774</td>
			<td >CD7 PE</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >333142</td>
			<td >CD11b PE</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >337899</td>
			<td >CD22 PE</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >345810</td>
			<td >CD56 PE</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >347222</td>
			<td >CD34 PERCP-CY5.5</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td>558714</td>
			<td>CD123 PERCP-CY5.5</td>
		</tr>
		<tr >
			<td ></td>
			<td >Beckman Coulter</td>
			<td>B49221</td>
			<td >CD117 PE-CY7</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td>562854</td>
			<td>CD33 BV421</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >345791</td>
			<td >CD19 APC</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >333143</td>
			<td >CD11b APC</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >345800</td>
			<td >CD33 APC</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td>345807</td>
			<td>CD38 APC</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >641411</td>
			<td >HLA-DR APC-H7</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td>560532</td>
			<td>CD44 APC-H7</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >562596</td>
			<td >CD13 BV421</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td>348811</td>
			<td>CD34 PE-CY7</td>
		</tr>
		<tr >
			<td ></td>
			<td >Beckman Coulter</td>
			<td>A96416</td>
			<td >CD45 Krome Orange</td>
		</tr>
		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td>655873</td>
			<td >CD45 HV500c</td>
		</tr>
		<tr >
			<td ></td>
			<td >BD Biosciences</td>
			<td >655051</td>
			<td>CST beads&#160;</td>
		</tr>
		<tr >
			<td ></td>
			<td >BD Biosciences</td>
			<td >555899</td>
			<td>Pharm lysing solution</td>
		</tr>
		<tr >
			<td ></td>
			<td >BD Biosciences</td>
			<td >644204</td>
			<td>Multicolor CompBeads</td>
		</tr>
		<tr >
			<td ></td>
			<td >BD Biosciences</td>
			<td >655051</td>
			<td>CST beads</td>
		</tr>
		<tr >
			<td ></td>
			<td >BD Biosciences</td>
			<td ></td>
			<td>FACSDiva software</td>
		</tr>
		<tr >
			<td ></td>
			<td >Cytognos</td>
			<td ></td>
			<td >Infinicyt&#160;</td>
		</tr>
		<tr >
			<td ></td>
			<td >Spherotech</td>
			<td>Euroflow RCP-30-5a</td>
			<td ></td>
		</tr>
		<tr >
			<td ></td>
			<td>Sigma-Aldrich</td>
			<td ></td>
			<td >Tuerk solution</td>
		</tr>
		<tr >
			<td ></td>
			<td >Spherotech</td>
			<td>BCP-100-5</td>
			<td>Blank Calibration Particles Beads</td>
		</tr>
		<tr >
			<td >&#160;&#160;</td>
			<td >&#160;&#160;</td>
			<td >&#160;&#160;</td>
			<td >HSA</td>
		</tr>
		<tr >
			<td >&#160;&#160;</td>
			<td >&#160;&#160;</td>
			<td >&#160;&#160;</td>
			<td >Azide</td>
		</tr>
		<tr >
			<td >&#160;&#160;</td>
			<td >&#160;&#160;</td>
			<td >&#160;&#160;</td>
			<td >ddH2O</td>
		</tr>
	</tbody>
</table>

comprehensive protocol to sample and process bone marrow for measuring measurable residual disease and leukemic stem cells in acute myeloid leukemia

急性髓细胞白血病 (AML) 的反应标准最近被重新建立, 通过形态学检查, 以确定患者是否已达到完全缓解 (CR)。由于骨髓中残留的 AML 细胞的生长, 进入 CR 的成年患者中约有一半将在12月内复发。这些剩余的白血病细胞的定量, 被称为最小或可测量的残留疾病 (MRD), 可以是一个强大的生物标志物预测这些复发。 此外, 对几项研究的回顾性分析表明, 在 AML 患者骨髓中, MRD 的存在与生存率的低下有关。不仅是白血病的总人数, 所反映的细胞与白血病相关的免疫表型 (LAIP), 与临床结果, 但也同样是不成熟的低频亚群白血病干细胞, 这两者都可以通过流式细胞仪或类似于 mrd 的方法进行监测。根据疾病特异性或与疾病相关的特征 (异常的分子标记或异常免疫表型) 检测残留白血病 (干细胞) 的敏感性化验有了显著的改善。在 AML。然而, 考虑到 AML 作为一种疾病的固有的异质性和复杂性, 在可能的情况下应协调骨髓取样方法和执行 MRD 和细胞分析法。在这篇手稿中, 我们描述了充足的骨髓穿刺取样, 运输, 样本处理的最佳多色流式细胞术评估的详细方法, 和门策略评估的 MRD 和细胞, 以帮助治疗决策为 AML 患者。

在90% 的 AML 患者中至少可以发现一个 LAIP。为了在诊断时生成原始爆炸细胞的 LAIP + 细胞的准确百分比, 必须在图 3中直观地确定爆破闸门的适当设置。在图 5中, 在 CD34+ 基元单元格中窝藏特定类型偏航的每个患者的示例都将被可视化。为在后续行动中测量 MRD, 早期定义的 LAIP + 细胞被门控, 并被定义为 LAIP + 细胞的百分比. 因此, 这门的设置是至关重要的, 以取得正确的 MRD 结果。图 6显示了在第二个循环诱导治疗后, 患者在完全缓解后复发, 患者在有了 MRD 阳性结果 (≥ 0.1%) 后反复发作。
目前的协议只适用于定义 CD34+ 细胞中的干细胞, 因为对这一舱室的深入描述表明, CD38 种群的干细胞, 如分数。为了在这一小部分中分离出 HSC, 使用额外的标记。通常选择的标记区分 CD45RA 和组合信道窝藏6个特定的, 标记为 PE 标记。根据进一步的临床评价, ≥0.004% 的截止水平被定义为 "内联" 阳性, 并与诊断中的生存率差有关, 而在以雷锋10中使用了0.0001% 的截止水平.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56386/56386fig1.jpg"> 图 1:骨髓涂片.A) 健康捐献者和 B) 反洗钱患者 (5 亚型)。姬姆萨染色显示在100x 倍放大。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56386/56386fig2.jpg"> 图 2: 正确的生物液体包装材料.关于运输生物液体的规则和条例的更多细节可以在疾病控制和预防中心的网站15中找到。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56386/56386fig3.jpg"> 图 3: 用于定义爆破单元的浇口策略.A) 门 CD45dim, B) 浇 CD34+ 爆炸。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56386/56386fig4.jpg"> 图 4: 门控淋巴细胞.A) 蓝细胞是在 FSC/SSC 图中显示的白血球, B) 绿色细胞是以 CD45高/SSC低为特征的淋巴细胞, C) 为 CD117 和 E) 负性的阴性的例子 CD13, F) 不同的细胞类型显示在一个概述人口树。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56386/56386fig5.jpg"> 图 5: LAIPs 在 AML 诊断中的应用.A) 基于十字血统偏航 (CD7 CD33 表达单元), B) LAIP 基于异步分化 (CD11b 表达细胞), C) CD13 基于表达比正常细胞 (LAIP非常高的 CD34 表达细胞), D)LAIP 基于 underexpression 比较正常细胞 (HLA-DR低在 CD33 表达细胞)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56386/56386fig6.jpg"> 图 6: LAIP 在治疗过程中遵循.a) LAIP 在诊断方面的定义 (CD34+/CD13+/CD33) 和在随访过程中 LAIP 在持续完全缓解、B) LAIP 诊断的定义 (CD117+/CD7+/CD33+) 和持续 LAIP 在患者随访中的连续性复发.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56386/56386fig7.jpg"> 图 7: 干细胞门控.A) 基于珠子的不同 CD34+/CD38 种群的门控 (CD38低位于珠子的上部边界下面, 而 CD38非常低被定义为代表该边缘的真实负单元格, 位于珠子的中点以下), B) 二根据 CD45RA阴性和 CD45RApos表达式, 在后续行动中区分 HSC 和 "内星" 的患者的例子。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充文件 1.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/Sampling_Form_MRD_1.pdf" target="_blank">请单击此处 downoad 此文件.</a>
补充文件 2.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/Sampling_Form_MRD_2.pdf" target="_blank">请单击此处 downoad 此文件.</a>
补充文件 3.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/Supplementary_information_revised_Cloos_et_al_final2018.docx" target="_blank">请单击此处 downoad 此文件.</a>

Watch this Scientific Journal Video about Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia at JoVE.c

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

检测微量或可测量残留疾病 (MRD) 是一种重要的预后标志物, 用于提炼风险评估和预测急性髓细胞白血病 (AML) 复发。这些全面的指导方针和建议采用最佳做法, 以一致和准确地识别和检测 MRD, 有助于作出有效的反洗钱治疗决定。

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

comprehensive-protocol-to-sample-process-bone-marrow-for-measuring

Nanyang Technological University

Research

JoVE Journal

Cancer Research

29.0K 次观看.  VU University Medical Center. 检测微量或可测量残留疾病 (MRD) 是一种重要的预后标志物, 用于提炼风险评估和预测急性髓细胞白血病 (AML) 复发。这些全面的指导方针和建议采用最佳做法, 以一致和准确地识别和检测 MRD, 有助于作出有效的反洗钱治疗决定。

视频: Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

详细的定性C1498小鼠细胞系及其关联白血病小鼠模型协议

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

科研

癌症研究

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

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

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

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

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 ...

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

离心淘洗制备不同细胞周期的原发性急性淋巴细胞白血病细胞

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

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

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

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

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

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. ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

流式细胞仪估测原发性急性髓系白血病和患者源性移植中白血病干细胞的诊断和随访

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a &#39;molecular timeline&#39; since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.

Herein, we present a protocol that details the technical aspects and essential requirements to ensure robust IG gene sequence analysis in patients with chronic lymphocytic leukemia (CLL), based on the accumulated experience of the European Research initiative on CLL (ERIC).

慢性淋巴细胞白血病免疫球蛋白基因序列分析: 从患者材料到序列解释

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

原发性白血病细胞代谢谱的评价

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

利用卡尔瓦里亚共同培养和教条法测量骨重塑和再造肿瘤-骨微环境

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

通过实体瘤移植植入骨模拟原发性骨肿瘤和骨转移

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...