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

この作品の一部、フランス語協会ローレット Fugain、LigueContre ルがん (北センター)、財団・ ド ・ フランス (白血病委員会)、SIRIC Oncolille、フランス国立がん研究所によって支えられました。

我々 は科学的な目的のための動物の使用のためのヨーロッパ規格に従ってください。本研究は、ローカル倫理委員会 (#3097-2015120414583482v3) によって承認されました。
1. サンプル準備
<ol><li>De novo AML 抗凝固エチレンジアミン四酢酸 (EDTA) を含む管内から骨髄の mL あたり 1.8 mg の濃度で骨髄 (2 mL) を収集します。</li>
	<li>続いて、赤血球および顆粒球、単核球を分離す?...

<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>

膜やフローサイトメトリーによる細胞質マーカーの正確かつ再現性のある評価は、機器の設定、毎日光配向、流体システム、光感度、標準化の適切な機能を確認が必要です。キャリブレーション ビーズを使用しています。
パラメトリック最適化を用いたフローサイトメトリー法による CD90 と CD45RA を使用して急性骨髄性白血病でブラスト セル人口の検出は、比較的単純?...

急性骨髄性白血病 (AML) は、白血病による死亡の最も一般的な原因です。当初、急性骨髄性白血病は造血幹細胞 (HSC) のクローン病分化、ブラスト細胞のそれに続く蓄積の逮捕によって特徴付けられる、造血機能の要素の生産量が減少します。最近では、ブラスト セル人口のクローンの不均一性の設置する本質的にシーケンス (NGS) の次世代戦略を使用して、腫瘍細胞1のクローン内の進化の存在を示します。
白血病幹細胞 (LSC) の存在する不均一性を拡張します。それは、この動的なセル区画は、白血病の進行と治療中に課せられた様々 な選択圧力を克服するために進化する仮定されます。したがって LSC は現在の化学療法レジメンに耐性があるし、深い臨床的意義2病気の再発を仲介になっています。様々 なマーカーは、ティム 36CD975や CLL 14CD1233LSC の特徴に記載されています。ブラスト細胞の cd 34 + CD38-コンパートメント LSC7豊かに言えますが、また通常 HSC が含まれています。Cd 34 + CD38 に適用される CD90 と CD45RA 式-(P6) コンパートメント (図 1) 正常および悪性造血前駆細胞8のいくつかの段階を分離することができます。具体的には、通常 cd 34 + CD38 CD90 + CD45RA HSCs、多能性前駆細胞 (MPP) cd 34 + CD38-CD90dimCD45RA-細胞など cd 34 + CD38-CD90-CD45RA + リンパ プライミング多能性前駆細胞 (LMPP) 細胞を急性骨髄性白血病の大半として定めることができる場合8 ,9。CD38 の平均蛍光強度 (MFI) は、CD34 陽性細胞コンパートメントの内で考慮することが大切です。CD38 強度その 3 つの新しい細胞のコンパートメントを定義するため: CD38 負 (P6)、CD38 dim (P7) と CD38 明るい (P8) (図 1)。CD38 の MFI は、これらの細胞は強く CD38 を特急として内部の肯定的な制御として hematogones およびプラズマ細胞のいずれかを使用して決定されるかもしれない。
免疫不全の合図/SCID/IL2Rγcnull (NSG) マウス モデルは通常の生着のため広く使用され悪性ひと造血細胞10,11。シリアル異種アッセイは、LSC または HSC 関数の実験的検証に使用されます。これらの研究は、大規模なシーケンスのアプローチによって広範囲に分析されています。それにもかかわらず、以下はその主な急性骨髄性白血病 (図 2) と比較して NSG マウスにしみ込んで AML ブラスト人口の LSC と HSC イムノフェノ タイプについて知られています。
LSC の番号を本質的に低、LSC の同定及び定量化が困難なここで説明する方法は、(残留 LSC として化学療法応答を評価するフローサイトメトリーアッセイ診断、臨床フォローとして使用する場合があります。急性骨髄性白血病再発を引き起こす)。LSC の存在は肯定的な微小残存病変 (MRD) 可能性があります。までに、MRD 分子法 (すなわちRT-PCR、NGS) に依存して急性骨髄性白血病で大抵監視10,12。しかし、このプロトコルでは我々 検出いくつかの HSC/LSC マーカー (CD34、CD38、CD45RA、CD90) の同時発現ヒト急性骨髄性白血病の一次爆発細胞に多重流れ cytometry2,8,9。この抗体の組み合わせ可能性があります任意の標準的な流れ cytometry 研究室で適用してこの流れ MRD の試金は、リアルタイムのポリメラーゼの連鎖反応 (RT-PCR) による MRD は不可能、特に興味深いすなわち場合、白血病特有の分子マーカーは、診断のアジア ・ マイル リミテッド サンプルで検出されません。さらに、このプロトコルはより敏感な分子 MRD 技術を補完するものそれは検出し、異常な造血前駆細胞を定量化することを目的として表します機能細胞特性 (図 3)。
臨床検査の大半で使用可能な抗体とフローサイトメトリーによる造血前駆細胞の 3 つの集団と成熟度が異なると推定される LSC コンパートメントを定量化する方法を示します。さらに、対応する患者-派生-異種移植片 (PDX) でこれらの細胞コンパートメントの存在を確認し、治療のフォロー アップ。

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			<td height="20" style="height:21px;width:363px;">Pancoll</td>
			<td style="width:189px;">PAN-Biotech</td>
			<td style="width:128px;">P04-60500</td>
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			<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>
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			<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>
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			<td>Gibco by life technologies</td>
			<td>14190-144</td>
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			<td height="20" style="height:21px;">AutoMACS Running Buffer &#8211; MACS Separation Buffer</td>
			<td>MACS Buffer Miltenybiotec</td>
			<td>130-091-221</td>
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			<td>Beckman Coulter</td>
			<td>PN IM0766U</td>
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			<td height="20" style="height:21px;">Anti-CD33-PC5.5 (clone D3HL60.251, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B36289</td>
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			<td height="20" style="height:21px;">Anti-CD90-APC (clone 5E10)</td>
			<td>Biolegend</td>
			<td>328114</td>
			<td style="width:467px;"></td>
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		<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>
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		<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>
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		<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>
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		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD45-KO (clone J.33, Iotest)</td>
			<td>Beckman Coulter</td>
			<td>B36294</td>
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			<td height="20" style="height:21px;">Anti-CD3-PE</td>
			<td>Beckman Coulter</td>
			<td>IM1451</td>
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			<td height="20" style="height:21px;">Anti-CD20-PE</td>
			<td>Beckman Coulter</td>
			<td>A07747</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:21px;">Anti-CD123-PC7</td>
			<td>Beckman Coulter</td>
			<td>B13647</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:21px;">Flow-Set</td>
			<td>Beckman Coulter</td>
			<td>A63492</td>
			<td style="width:467px;"></td>
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		<tr height="20">
			<td height="20" style="height:21px;">Flow-Check</td>
			<td>Beckman Coulter</td>
			<td>A63493</td>
			<td style="width:467px;"></td>
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		<tr height="20">
			<td height="20" style="height:21px;">Navios</td>
			<td>Beckman Coulter</td>
			<td>DS-14644A</td>
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			<td height="20" style="height:21px;">LSR Fortessa X20</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:21px;">CST BD</td>
			<td>BD Biosciences</td>
			<td>655051</td>
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			<td height="20" style="height:21px;">FacsFlow&#160;</td>
			<td>BD Biosciences</td>
			<td>336911</td>
			<td></td>
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		<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>
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flow cytometry to estimate leukemia stem cells in primary acute myeloid leukemia and in patient-derived-xenografts, at diagnosis and follow up

急性骨髄性白血病 (AML) は異質と致命的な病気を治療されていない場合。成人の白血病による死亡の最も一般的な原因です。当初、急性骨髄性白血病は造血幹細胞 (HSC) の病気差別化、爆発性白血病細胞のそれに続く蓄積の逮捕によって特徴付けられる、造血の機能要素の生産量が減少します。不均一性白血病幹細胞 (LSC) の存在まで、この動的セル白血病の進行と治療中に課せコンパートメントは様々 な選択圧力を克服するために進化しています。LSC 人口をさらに定義するには、CD90 と CD45RA は cd 34 + CD38-細胞コンパートメント内正常造血多能性前駆細胞の差別をことができます。ここでは、多重フローサイトメトリーを用いたヒト急性骨髄性白血病の一次爆発細胞にいくつかの推定 LSC マーカー (CD34、CD38、CD45RA、CD90) の同時発現を検出するためのプロトコルをまとめました。さらに、3 つの前駆細胞集団と増加する成熟の程度と推定される LSC 人口を定量化する方法を示します。対応する患者-派生-異種移植片におけるこれらの個体群の存在を確認しました。検出のこのメソッドおよび推定 LSC の定量化が化学療法応答の臨床フォロー アップに使用する (すなわち。、微少残存病変)、残留 LSC は急性骨髄性白血病再発を引き起こす可能性があります。

正常および悪性ひと骨髄サンプルで前駆細胞集団を決定する方法をご紹介します。成功 PDX から骨髄および脾臓を収集し、上記のように多重フローサイトメトリーを実行.ブラスト細胞診断患者検体と対応する PDX cd 34 + CD38-前駆コンパートメントでは特に、特に推定 LSC の濃縮のいくつかの集団を比較しました。PDX 診断患者サンプル (0.53%、図 2、 D対 3.48%) と?...

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

フローサイトメトリーによる主な急性骨髄性白血病細胞白血病幹細胞マーカーの同時発現を検出するためのプロトコルの概要を説明します。3 前駆細胞集団と増加する成熟の程度と推定される LSC 人口を定量化する方法を示します。対応する患者-派生-異種移植片におけるこれらの個体群の存在を確認しました。

フローサイトメトリーを診断とフォローで主に急性骨髄性白血病、白血病幹細胞患者由来異種での見積もりに

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

サンプルおよび測定可能な残存病変と急性骨髄性白血病の白血病幹細胞を測定するプロセス骨髄に対する包括的なプロトコル

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

がん研究

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

遠心浄化によって異なった細胞周期の段階で主に急性リンパ性白血病細胞の調製

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

肉腫患者由来異種移植片からの腫瘍始発細胞の単離と特徴付け

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

慢性リンパ性白血病の新規 NFAT2 ターゲット遺伝子を識別するクロマチン免疫沈降法

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

主な白血病細胞の代謝プロファイルの評価

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

マウス造血幹細胞および前駆細胞およびMLL-AF9駆動白血病におけるミトコンドリア活性酸素種のフローサイトメトリック解析

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

核/細胞質タンパク質の密売を監視する原発性慢性リンパ球性白血病細胞の細胞分画

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

構造DNA改変解析と患者由来異種移植片を用いたがんの標的治療の試験

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

急性骨髄性白血病におけるバルク亜集団と幹細胞を区別するNKG2Dリガンド表面検出の2つのフローサイトメトリックアプローチ

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

マウスにおける急性骨髄性白血病発症のための腹腔内移植

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