Name: flow cytometry to estimate leukemia stem cells in primary acute myeloid leukemia and in patient-derived-xenografts, at diagnosis and follow up
Uploaded: 2018-03-26T13:00:00
Description: Watch this Scientific Journal Video about Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up at JoVE.com

这项工作部分是由法国协会 Laurette Fugain、LigueContre 乐癌症 (北中心)、法国基金会 (白血病委员会)、SIRIC Oncolille 和法国国家癌症研究所支持的。

我们遵循欧洲为科学目的使用动物的标准。这项研究是由地方伦理委员会 (#3097 2015120414583482v3) 批准的。
1. 样品准备
<ol><li>以1.8 毫克每毫升骨髓的浓度为单位, 在含有抗凝血乙二甲酸 (EDTA) 的管中收集骨髓 (2 毫升). </li>
	<li>进行 Ficoll 分离, 密度梯度分离, 以隔离单个核细胞从红细胞和粒粒, 如下所为。稀释骨髓在3容量磷酸盐缓冲盐水 (PBS: 氯化钠137毫米, 氯化钾2.7 毫米,...

<ol>
	<li>Shlush, L. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+the+origins+of+relapse+in+acute+myeloid+leukaemia+to+stem+cells.">Tracing the origins of relapse in acute myeloid leukaemia to stem cells.</a> Nature. , (2017).</li><li>Terwijn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemic+stem+cell+frequency:+a+strong+biomarker+for+clinical+outcome+in+acute+myeloid+leukemia.">Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.</a> PLoS One. 9 (9), e107587 (2014).</li><li>Jordan, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interleukin-3+receptor+alpha+chain+is+a+unique+marker+for+human+acute+myelogenous+leukemia+stem+cells.">The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.</a> Leukemia. 14 (10), 1777-1784 (2000).</li><li>van Rhenen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+novel+AML+stem+cell+associated+antigen+CLL-1+aids+in+discrimination+between+normal+and+leukemic+stem+cells.">The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.</a> Blood. 110 (7), 2659-2666 (2007).</li><li>Bonardi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proteomics+and+transcriptomics+approach+to+identify+leukemic+stem+cell+(LSC)+markers.">A proteomics and transcriptomics approach to identify leukemic stem cell (LSC) markers.</a> Mol Cell Proteomics. 12 (3), 626-637 (2013).</li><li>Kikushige, Y., Miyamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+TIM-3+as+a+Leukemic+Stem+Cell+Surface+Molecule+in+Primary+Acute+Myeloid+Leukemia.">Identification of TIM-3 as a Leukemic Stem Cell Surface Molecule in Primary Acute Myeloid Leukemia.</a> Oncology. 89, 28-32 (2015).</li><li>Bonnet, D., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+acute+myeloid+leukemia+is+organized+as+a+hierarchy+that+originates+from+a+primitive+hematopoietic+cell.">Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.</a> Nat Med. 3 (7), 730-737 (1997).</li><li>Kersten, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD45RA,+a+specific+marker+for+leukaemia+stem+cell+sub-populations+in+acute+myeloid+leukaemia.">CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia.</a> Brit J Haematol. 173 (2), 219-235 (2016).</li><li>Goardon, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+LMPP-like+and+GMP-like+leukemia+stem+cells+in+acute+myeloid+leukemia.">Coexistence of LMPP-like and GMP-like leukemia stem cells in acute myeloid leukemia.</a> Cancer Cell. 19 (1), 138-152 (2011).</li><li>Ng, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+17-gene+stemness+score+for+rapid+determination+of+risk+in+acute+leukaemia.">A 17-gene stemness score for rapid determination of risk in acute leukaemia.</a> Nature. 540 (7633), 433-437 (2016).</li><li>Shultz, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lymphoid+and+myeloid+cell+development+in+NOD/LtSz-scid+IL2R+gamma+null+mice+engrafted+with+mobilized+human+hemopoietic+stem+cells.">Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.</a> J. Immunol. 174 (10), 6477-6489 (2005).</li><li>Roloff, G. W., Lai, C., Hourigan, C. S., Dillon, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+Advances+in+the+Measurement+of+Residual+Disease+in+Acute+Myeloid+Leukemia.">Technical Advances in the Measurement of Residual Disease in Acute Myeloid Leukemia.</a> J Clin Med. 6 (9), (2017).</li><li>Swamydas, S., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Purification+and+Labeling+of+Mouse+Bone+Marrow+Neutrophils+for+Functional+Studies+and+Adoptive+Transfer+Experiments.">Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments.</a> J Vis Exp. (77), e50586 (2013).</li><li>Gabert, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+and+quality+control+studies+of+'real-time'+quantitative+reverse+transcriptase+polymerase+chain+reaction+of+fusion+gene+transcripts+for+residual+disease+detection+in+leukemia+-+a+Europe+Against+Cancer+program.">Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program.</a> Leukemia. 17 (12), 2318-2357 (2003).</li></ol>

通过流式细胞术对膜或细胞质标记进行准确和可重复的评估, 要求仪器设置每天都要经过验证, 以进行光学对准、射流系统的正常运行、光学灵敏度和标准化使用校准珠。
应用多参数流式细胞仪分析 CD90 和 CD45RA 检测 AML 中的爆炸细胞群是一种比较简单可靠的方法。这项分析的一个关键步骤是准确评估 CD38 在爆炸细胞中的表达, 以便正确地进行 CD34+CD38 的门控。CD38 强度的定义是...

急性髓系白血病 (AML) 是导致白血病相关死亡的最常见原因。首先, AML 是一种造血干细胞 (HSC) 的克隆病, 其特点是: 停止分化, 随后累积爆炸细胞, 减少功能性造血元件的生产。近年来, 通过使用下一代测序策略和显示肿瘤细胞内克隆进化的存在性, 建立了爆炸细胞种群的无性系异质性, 并表明其存在于1。
异质性延伸到白血病干细胞的存在。据推测, 这种动态细胞室的演变, 以克服各种选择压力强加在白血病的进展和治疗。因此, 对目前的化疗方案和介导的疾病复发有抵抗力, 其临床意义2。各种标记都被描述为特征 CD1233、CLL-14、CD975或 TIM-36等。CD34+CD38 的爆破细胞的隔室被认为是丰富的, 为7 , 但也包括正常的 HSC。CD90 和 CD45RA 表达式应用于 CD34+CD38 (P6) 隔间 (图 1) 允许隔离正常和恶性造血前体的几个阶段8。具体地说, 正常的 CD34+CD38-CD90+CD45RA-HSCs, 多能祖 (MPP) 像 CD34+CD38-CD90dimCD45RA 细胞和 CD34+CD38-CD90-CD45RA+ 淋巴引物多能祖 (LMPP) 细胞可以确定在大多数 AML 病例中,8 ,9。CD38 的平均荧光强度 (多边基金会) 在 CD34+ 细胞室中尤为重要。因为 CD38 强度定义了三个新的细胞间隔: CD38 阴性 (P6), CD38 暗淡 (P7) 和 CD38 明亮 (P8) (图 1)。CD38 可以通过 hematogones 和/或血浆细胞作为内部阳性控制来确定, 因为这些细胞强烈表达 CD38。
immunodeficient NOD/SCID/IL2Rγcnull (核供应集团) 鼠标模型广泛用于植入正常和恶性人类造血细胞10,11。采用系列异种移植方法对功能进行实验验证。通过大规模测序方法对这些研究进行了广泛的分析。然而, 与其主要的 aml (图 2) 相比, 反洗钱爆炸种群的嫁接和 HSC 免疫表型对核供应的小鼠的认识较少。
该方法的数量在本质上是低的, 因此, 识别和量化的研究是有挑战性的, 这里描述的方式可以作为一个流细胞分析诊断和临床随访评估化疗反应 (作为残留的可能引起 AML 复发)。存在的可显示阳性的最小残留疾病 (MRD)。迄今为止, 在反洗钱中的 MRD 监测主要依靠分子方法 (即, rt-pcr,)10,12。然而, 在本协议中, 我们通过多参数流式细胞术 2, 8 9, 在人类反洗钱的主要爆炸细胞中检测出几种 HSC/CD34 标记 (CD38、CD45RA、CD90) 的同时蛋白表达.这种抗体组合可应用于任何标准流式细胞仪实验室, 这种流动的 mrd 检测特别有趣, 当 MRD 的实时聚合酶链反应 (RT PCR) 是不可能的,即, 如果白血病特异分子标记在诊断 AML 样本中无法检测到。此外, 本议定书补充了更为敏感的分子 MRD 技术, 因为它旨在检测和量化代表功能细胞特征的异常造血祖细胞 (图 3)。
我们展示了如何用流式细胞术来量化三个造血祖群和不同成熟度的可供选择的隔间室, 而大多数临床实验室都有抗体。此外, 我们证实了这些细胞室在相应的病人派生移植 (PDX) 和治疗随访。

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	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:363px;">Pancoll</td>
			<td style="width:189px;">PAN-Biotech</td>
			<td style="width:128px;">P04-60500</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="75">
			<td height="75" style="height:75px;">RBC Lysis Buffer (10x)</td>
			<td>Ozyme</td>
			<td>BLE420301</td>
			<td style="width:467px;">RBC Lysis solution A : 8.29 g of NH4Cl + 0.037 g of Na2EDTA in 100 mL of sterile H2O and RBC Lysis solution B: 1 g of KHCO3 in 100 mL of sterile H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">NOD/SCID/IL2R&#947;cnull&#160; (NSG)&#160; mice</td>
			<td>JACKSON LABORATORY</td>
			<td>Stock No: 005557</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">PBS</td>
			<td>Gibco by life technologies</td>
			<td>14190-144</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">AutoMACS Running Buffer &#8211; MACS Separation Buffer</td>
			<td>MACS Buffer Miltenybiotec</td>
			<td>130-091-221</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD36-FITC (clone FA6-152, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>PN IM0766U</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD33-PC5.5 (clone D3HL60.251, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B36289</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD90-APC (clone 5E10)</td>
			<td>Biolegend</td>
			<td>328114</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD19-ECD (clone J3-119, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>A07770</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD34-AA700 (clone 581, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B92417</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD45RA-APC-H7 (clone HI100)&#160;</td>
			<td>Beckton Dickinson</td>
			<td>560674</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD38-Pacific Blue (clone LSI98-4-3, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B92396</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD45-KO (clone J.33, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B36294</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD3-PE</td>
			<td>Beckman Coulter</td>
			<td>IM1451</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD20-PE</td>
			<td>Beckman Coulter</td>
			<td>A07747</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD123-PC7</td>
			<td>Beckman Coulter</td>
			<td>B13647</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Flow-Set</td>
			<td>Beckman Coulter</td>
			<td>A63492</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Flow-Check</td>
			<td>Beckman Coulter</td>
			<td>A63493</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Navios</td>
			<td>Beckman Coulter</td>
			<td>DS-14644A</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">LSR Fortessa X20</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">CST BD</td>
			<td>BD Biosciences</td>
			<td>655051</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">FacsFlow&#160;</td>
			<td>BD Biosciences</td>
			<td>336911</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-hCD45-FITC</td>
			<td>Biolegend</td>
			<td>BLE304006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Anti-mCD45-APC</td>
			<td>Biolegend</td>
			<td>BLE103112</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">EasySep human PE selection kit</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">EasySep&#160; magnet&#160;</td>
			<td>Stem Cell Technologies</td>
			<td>18551</td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometry to estimate leukemia stem cells in primary acute myeloid leukemia and in patient-derived-xenografts, at diagnosis and follow up

急性髓系白血病 (AML) 是一种异种, 如果不治疗, 致命的疾病。这是成人白血病相关死亡的最常见原因。首先, AML 是一种造血干细胞 (HSC) 的疾病, 其特点是: 停止分化, 随后聚集白血病爆炸细胞, 减少功能性造血元件的生产。异质性延伸到白血病干细胞的存在, 这一动态细胞室的演变, 以克服各种选择压力强加在白血病的进展和治疗。为了进一步定义非定常种群, 添加 CD90 和 CD45RA 允许在 CD34+CD38 细胞间区分正常的 HSCs 和多能祖。在这里, 我们概述了一个协议, 以检测几个假定的 CD34, CD38, CD45RA, CD90) 的人反洗钱的主要爆炸细胞, 多参数流式细胞术的同时表达。此外, 我们还展示了如何量化三个祖群体和一个假定的多项种群的成熟程度。我们证实了这些人群存在于相应的病人衍生的移植。这种检测方法和定量的假定的可用于临床随访的化疗反应 (i. e., 最小残留疾病), 因为残端细胞可能导致 AML 复发。

在这里, 我们提出了一个方法来确定祖细胞的正常和恶性人骨髓标本。我们从成功的 PDX 收集骨髓和脾脏, 并进行多参数流式细胞术, 如上文所述。我们比较了在诊断病人样本和相应的 PDX 之间的几个亚群, 特别是 CD34+CD38 祖室, 特别是丰富的假定的。我们发现, 与诊断病人样本相比, PDX 的 CD34+CD38 爆炸分数较高 (3.48% vs 0.53%,图 2C, D)。有趣的是, 我们发现在 PDX 中, 与主...

Watch this Scientific Journal Video about Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up at JoVE.com

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

通过流式细胞术, 我们勾勒出一种检测白血病干细胞标记物同时表达的协议。我们展示了如何量化三个祖群体和一个假定的多项种群的成熟程度。我们证实了这些人群存在于相应的病人衍生的移植。

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 ...

The overall goal of this procedure is to estimate leukemia stem cells in acute myeloid leukemia and in patient-derived-xenografts by flow cytometry at diagnosis and at treatment follow up. This method can answer key questions in the hematology/oncology field, such as the clinical relevance of the quantitative assessment of leukemia stem cells by flow cytometry in acute myeloid leukemia. The main advantage of this technique is that it is applicable to most hematology/oncology laboratories with commonly used antibodies.This procedure will be demonstrated by Thomas, a post-doc, Fanny, a grad student, and Adeline, an engineer from my laboratory. To begin, collect two milliliters of bone marrow from de novo AML in tubes containing 1.8 milligrams per milliliter of the anti-coagulant EDTA. Isolate mononuclear cells from red blood cells and granulocytes by first diluting bone marrow in three volumes of PBS.Then, carefully overlay one volume of ficol with one volume of diluted bone marrow. Spin the density gradient at 300g for 30 minutes without a break. Then, transfer the white buffy coat layer of mononucleated cells into a new sterile tube.To remove the supernatants contaminating serum components, add 10 milliliters of PBS and centrifuge the cells at 300g for five minutes before repeating the wash. Lyse the red blood cells by adding two milliliters of lysis buffer to the cell pellet and gently vortex. Then, incubate the cells at room temperature for five minutes.Centrifuge the cells at 300g for five minutes and remove the supernatant. Then, use PBS to wash the cells. After isolating blast cells from bone marrow according to the text protocol, under a laminar flow hood, insert an unconditioned NSG mouse into a restraining device and inject five times 10 to the sixth AML blast cells into the tail vein.Return the mouse to an individual ventilated cage under conventional and specific pathogen-free conditions. Every two weeks, use a submandibular collection method with a hematocrit capillary to draw 60 microliters of blood. Transfer the capillary into a five milliliter tube then with a sterile gauze pad, apply gentle pressure to stop the bleeding.Add one milliliter of lysis buffer to the tube and gently vortex. Then, incubate the cells at room temperature for five minutes. Centrifuge the cells at 300g for five minutes.Then, after removing the supernatant, add 100 microliters of PBS with 10%BSA, three microliters of anti-human CD45 FITC, incubate for 30 minutes. Then, wash the cell pellet two times in PBS, two milliliters, with 10%BSA and centrifuge at 300g for five minutes. Every two weeks, use flow cytometry to analyze the engraftment by chimerism analysis of human versus murine CD45 expression.When the blast count is greater than 70%or severe clinical signs of sickness are observed, sacrifice the animal and collect mononuclear blast cells from a crushed spleen and from bone marrow by using PBS to flush tibias and femurs as previously described. To carry out panel staining, combine conjugated antibodies with human primary or patient-derived-xenograft, or PDX samples, and vortex the tubes. Then, incubate the cells in the dark at room temperature for 15 to 30 minutes.Remove the unbound antibodies by using two milliliters of PBS to wash the cells. Re-suspend the cells in 450 microliters of PBS, then proceed to flow cytometry analysis and MRD monitoring. To accurately and reproducibly assess membrane of cytoplasmic markers by flow cytometry, verify optical alignment.Proper tuning of the 3D system and optical sensitivity and stomatization using stomazine calibration beats every day. Use normal bone marrow to set the gates for CD38. A minimum of 500, 000 events are required and it is verified that each flow run is regular and homogenous.In addition, doublets, as well as dead cells and cell debris are eliminated. Immature hematopoietic cells are defined by being CD45 intermediate, SSC low. And the CD38 positive control or hematogones, are CD19/CD38 double positive.Then P8 or CD38 positive, P7 or CD38 intermediate, and P6 or CD38 negative are defined. The P6 cells can be further subdivided into HSC, MPP, and LSC. Since it is a normal bone marrow it is LSC negative.Using multiparametric flow cytometry, it was found that the CD34 positive, CD38 negative blast fraction was higher in PDX compared to the diagnostic patient sample. Interestingly, a higher percentage of putative LSC was found in the PDX compared to the primary patient sample, suggesting a possible enrichment of malignant progenitor cells in the hematologic tissues in this mouse model. Upon comparison of the blast progenitor populations in two different patient samples, the CD34 positive, CD38 negative fraction increased from 0.06%at diagnosis to 3.33%at the first treatment follow-up in the first patient.In contrast, the CD34 positive, CD38 negative fraction decreased in the second patient from 7.8%at diagnosis to 2.27%at the first treatment follow-up. Accordingly, the putative LSC fraction increased in the first patient from 0%at diagnosis to 0.65%and decreased in the second patient from 6.57%at diagnosis to 1.44%at treatment follow-up. Minimal residual disease or MRD monitoring, using as molecular markers the NPM1 mutation in patient one and the CBF-beta MYH11 gene fusion for patient two showed that molecular MRD decreased less in the first patient compared to the second patient for the first treatment follow-up point.Once mastered, this technique can be done in two hours if it is performed properly. While you're attempting this procedure, it is important to verify flow cytometric settings and CD38 gauging. Following this procedure, additional methods can be performed, like flow cytometric cell sorting in order to answer additional questions like functional leukemia stem cell assess.After watching this video you should have a good understanding of how to detect and quantify leukemia stem cells in patient samples in patient-derived-xenografts. Don't forget that working with patient samples can be hazardous and precautions such as GOP guidelines and institutional safety procedures should always be taken while you are performing this procedure.

Research

JoVE Journal

Cancer Research

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

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 ...

科研

癌症研究

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 ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

一种检测慢性淋巴细胞白血病新 nfat2 靶基因的染色质免疫沉淀法

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

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 ...

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 ...

Subcellular Fractionation of Primary Chronic Lymphocytic Leukemia Cells to Monitor Nuclear/Cytoplasmic Protein Trafficking

原发性慢性淋巴细胞白血病细胞的细胞次细胞分段监测核/细胞质蛋白贩运

Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

使用结构DNA改变分析和患者衍生异种移植测试癌症的靶向疗法

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

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 ...