Name: comprehensive protocol to sample and process bone marrow for measuring measurable residual disease and leukemic stem cells in acute myeloid leukemia
Uploaded: 2018-03-05T12:30:00
Duration: 9 min 57 s
Description: 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

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 >120995496,060996589 and 013729</td>
			<td >FACS Canto II&#160;</td>
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		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >555360</td>
			<td>CD7 FITC</td>
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			<td >DAKO</td>
			<td >F076701</td>
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			<td >Sanquin</td>
			<td >M1613</td>
			<td>CD36 FITC</td>
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		<tr >
			<td ></td>
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			<td >332778</td>
			<td>CD15 FITC</td>
		</tr>
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			<td ></td>
			<td>BD Biosciences</td>
			<td>335039</td>
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			<td >130-080-801</td>
			<td >CD133 PE</td>
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			<td>562566</td>
			<td>Clec12a PE</td>
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			<td>BD Biosciences</td>
			<td>565568</td>
			<td>Tim-3 PE</td>
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			<td >332774</td>
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			<td>BD Biosciences</td>
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			<td>BD Biosciences</td>
			<td >337899</td>
			<td >CD22 PE</td>
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			<td>BD Biosciences</td>
			<td >345810</td>
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			<td ></td>
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			<td ></td>
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			<td>CD123 PERCP-CY5.5</td>
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			<td ></td>
			<td >Beckman Coulter</td>
			<td>B49221</td>
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			<td ></td>
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			<td>562854</td>
			<td>CD33 BV421</td>
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		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td >345791</td>
			<td >CD19 APC</td>
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			<td ></td>
			<td>BD Biosciences</td>
			<td >333143</td>
			<td >CD11b APC</td>
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			<td ></td>
			<td>BD Biosciences</td>
			<td >345800</td>
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			<td ></td>
			<td>BD Biosciences</td>
			<td>345807</td>
			<td>CD38 APC</td>
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			<td ></td>
			<td>BD Biosciences</td>
			<td >641411</td>
			<td >HLA-DR APC-H7</td>
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		<tr >
			<td ></td>
			<td>BD Biosciences</td>
			<td>560532</td>
			<td>CD44 APC-H7</td>
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			<td ></td>
			<td>BD Biosciences</td>
			<td >562596</td>
			<td >CD13 BV421</td>
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		<tr >
			<td ></td>
			<td>BD Biosciences</td>
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		<tr >
			<td ></td>
			<td >Beckman Coulter</td>
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			<td ></td>
			<td>BD Biosciences</td>
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			<td >BD Biosciences</td>
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			<td>Pharm lysing solution</td>
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			<td ></td>
			<td >BD Biosciences</td>
			<td >644204</td>
			<td>Multicolor CompBeads</td>
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		<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>
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			<td >&#160;&#160;</td>
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			<td >&#160;&#160;</td>
			<td >Azide</td>
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			<td >&#160;&#160;</td>
			<td >&#160;&#160;</td>
			<td >&#160;&#160;</td>
			<td >ddH2O</td>
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	</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/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/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/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/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/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/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/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/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/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 ...

The overall goal of this protocol is to perform an accurate testing of bone marrow samples from acute myeloid leukemia patients for measurable residual disease including the quantification of leukemia stem cells. This method can provide an accurate assessment of measurable residual disease in bone marrow samples of acute myeloid leukemia patients. The main advantage of this approach is that a close following of the protocol yields highly-reproducible, high-quality results.The implications of this technique extends toward clinical decision-making as it can be used as a read-out of patients'response to treatment. Demonstrating the procedures will be Jeroen Janssen, hematologist, Gerrit Sijm, technician of the diagnostic hematology laboratory and Jennifer Scheick and Sander Snel, technicians of the MRD team. With the patient in the lateral decubitus position, mark the superior posterior iliac spine with a pen and disinfect the skin of the intended biopsy area with 0.5 to 1%chlorhexidine in ethanol in an outward circular motion.After administering a local anesthetic, pick up a 15-gauge 2.8-inch needle with the proximal end in the palm and the index finger against the side of the metal shaft of the needle near the tip for control. Using gentle but firm pressure, introduce the needle with a quick alternating pronating supinating movement through the anesthetized skin toward the iliac spine. When the needle comes into contact with the posterior iliac spine, remove the stilette and attach an empty 10-milliliter syringe to the needle.Gently withdraw the plunger to create negative pressure within the syringe until up to 10 milliliters of bone marrow spicules have been harvested. Do not take more than 10 mils of bone marrow per aspirate to avoid hemodilution. Remove the syringe and place the stilette back onto the needle.Then, eject about two milliliters of the marrow into a watch glass and ensure that enough sample is drawn to be injected into a heparin-coated eight-milliliter tube and immediately invert the tube. To prevent clotting, it is important to invest the anticoagulant-containing tube as soon as the bone marrow is added. For optimal morphological assessment, use a plastic spatula to transfer spicules from the aspirate in the watch glass onto a glass slide, and carefully glide another glass slide on top of the marrow.After drying, stain the spicules with May-Grunwald Giemsa for analysis by light microscopy. Place the bone marrow tube in a plastic holder and place the holder into a plastic container with an ambient gel pack and a completed request form. Before analyzing the bone marrow by flow cytometry, start up the flow cytometer and open the flow cytometry analysis software to create a new experiment with a forward scatter versus side scatter dot plot.For assessment of the size and granularity of the cells, load unlabeled lysed peripheral blood cells from a healthy donor and select the Acquire Cells function. Gate the lymphocytes and adjust the forward and side scatter voltages to reach the appropriate mean target values for the gated cell population. Then, acquire data for at least 10, 000 events.To set up the target florescence channel photo multiplier tube voltages, first use eight-peak rainbow bead calibration particles according to the manufacturer's instructions to acquire 10, 000 events at a low flow rate. Gate the singlet beads in the forward versus side scatter dot plot followed by gating the 7th peak in the FITC-PE dot plot. Gate the resulting bead populations for each fluorophore.Continue the acquisition of the eight-peak rainbow bead suspension and adjust and fine-tune PMT voltages in all fluorescence channels to reach target MFI values according to the manufacturer's instructions. Use Record to collect data of about 2, 000 events and check the MFI for the 7th peak beads. Next, add a sufficient volume of each experimental flourochrome conjugated antibody to stain the beads in the corresponding florescence tubes minus one control and vortex the tubes thoroughly.Create a new blank experiment with the same compensation parameters taking care that the forward scatter threshold is set to 5, 000 and the compensation values of all the flourochromes are zero. To create the compensation controls, select the Create Compensation Control function and load the unstained control tube. Adjust the P1 gate around the singlet bead population and confirm that the P1 gate contains only singlet beads.Right-click the P1 gate and select Apply to All Compensation Controls. Then, record data for all of the single labeled florescence tubes confirming that the P2 gate encompasses the positive population on each florescence histogram. To stain the cells for flow cytometric analysis, transport the bone marrow to the laboratory, check the request form to verify patient study number and date of birth and determine what needs to be stained.For MRD, we use a staining panel consisting of four tubes. Here we demonstrate the staining of the LSC panel which consists of one tube. After cell count, transfer the appropriate volume of bone marrow into a new 15-milliliter tube.Add lysis buffer at 10 times the experimental cell volume to lyse the red blood cells. Invert the tube to mix and incubate the cells for 10 minutes at room temperature. At the end of the lysis period, pellet the cells by centrifugation.Resuspend the cells in 15 milliliters of wash buffer at room temperature and harvest the cell pellet again by centrifugation. In the meantime, pipette the appropriate amount of antibody cocktail solution in five-milliliter FACS tubes. Shown here is the panel for the stem cell tube.Resuspend the pellet in wash buffer and add the cell's suspension to the FACS tube containing the monoclonal antibody cocktail solution for a 15-minute incubation in the dark. Then, wash the cells in wash buffer and pellet the cells by centrifugation. After centrifugation, resuspend the pellet in 400 microliters of fresh wash buffer.To analyze the leukemic stem cells, add four microliters of blank beads to the cell suspension that is stained with the stem cell antibody cocktail. Then, read the samples using the previously set parameters acquiring at least four times 10 to the 6th events from the experimental patient samples. To identify the leukemia-associated immunophenotype observed in approximately 90%of acute myeloid leukemia patients, it is crucial that the blast gates are set appropriately as illustrated.Here, examples of data from individual patients harboring different types of aberrancy on the CD34-positive primitive cells are shown. In these graphs, a patient who remains in complete remission and a patient who relapsed after having measurable residual disease positive results after the second cycle of induction therapy can be observed underscoring the need for accurate gate setting prior to sample analysis. The identification and quantification of LSC requires additional analysis of aberrancies specifically on CD34-positive CD38-negative blast cells as shown in these graphs.While performing this protocol, it's very important to have specialized and backup personnel for the bone marrow sampling, the transport, the experimental work and analysis steps of this procedure. This procedure can also be used to perform cytogenetic and molecular analysis to answer additional questions about the correlation between specific cell populations and molecular aberrancies of interest, or to refine the risk group of a patient for predicting clinical outcome. After its development, this technique paved the way for researchers in the field of hematology to explore residual disease in AML patients after treatment.After watching this video, you should have a good understanding how we accurately identify and quantify residual disease by using flow cytometry including the collection and transportation of bone marrow samples of AML patients.

Research

JoVE Journal

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详细的定性C1498小鼠细胞系及其关联白血病小鼠模型协议

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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

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 ...

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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. ...

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

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 ...

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

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

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

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 ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

NKG2D 利甘德表面检测的两种流细胞学方法，以区分干细胞与急性骨髓性白血病中的散装亚群

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

腹膜内移植在小鼠中产生急性髓系白血病

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...