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

Dette arbejde blev delvis støttet af franske Association Laurette Fugain, LigueContre le kræft (North Center), Fondation de France (leukæmi Udvalget), SIRIC Oncolille og fransk National Cancer Institute.

Vi følger europæiske normer for anvendelse af dyr til videnskabelige formål. Denne undersøgelse blev godkendt af det lokale etiske udvalg (#3097-2015120414583482v3).
1. Prøvetilberedning
<ol><li>Indsamle knoglemarv (2 mL) fra de novo AML i rør indeholdende antikoagulerende ethylendiamintetra syre (EDTA) ved en koncentration på 1,8 mg pr. mL af knoglemarven.</li>
	<li>Udføre Ficoll separation, en befolkningstæthed gradient adskillelse at isolere mononukleære celler fra rød...

<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. 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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>

Nøjagtige og reproducerbare vurdering af membran eller cytoplasmatisk markører ved flowcytometri kræver at Apparatindstillingen er efterprøvet hver dag for optisk tilpasningen, korrekt funktion af det fluidic system, optisk følsomhed og standardisering ved hjælp af kalibrering perler.
Påvisning af blast cellepopulationer i AML med CD90 og CD45RA ved multi parametrisk flow flowcytometri analyse er en relativt enkel og pålidelig metode. Et afgørende skridt i denne analyse er en nøjagti...

Akut myeloid leukæmi (AML) er den mest almindelige årsag til leukæmi-associerede dødelighed. I første omgang er AML en klonal sygdom af hæmatopoietisk stamceller (HSC) karakteriseret ved anholdelsen af differentiering, efterfølgende ophobning af blast celler, og reduceret produktion af funktionelle hæmatopoietisk elementer. For nylig, klonede heterogenitet af blast celle befolkning er blevet etableret med det væsentlige ved hjælp af næste generation sequencing (NGS) strategier og viser eksistensen af intra klonal evolution af tumor celler1.
Heterogenitet strækker sig til tilstedeværelsen af leukemic stamceller (LSC). Det er en hypotese, denne dynamiske celle rum udvikler sig for at overvinde forskellige udvalg pres pålagt under leukæmi progression og behandling. Derfor LSC formodes at være modstandsdygtige over for aktuelle kemoterapeutiske regimer og mægle sygdom tilbagefald, med dybtgående kliniske konsekvenser2. Forskellige markører er blevet beskrevet til at karakterisere LSC som CD1233, CLL-14, CD975 eller TIM-36. CD34 + CD38-rum af blast celler anses for at være beriget til LSC7 men også omfatter normale HSC. CD90 og CD45RA udtryk anvendes til CD34 + CD38-(P6) rum (figur 1) tillader for at adskille flere stadier af normale, og ondartet hæmatopoietisk prækursorer8. Specifikt, normal CD34 + CD38-CD90 + CD45RA-HSCs, Multipotente stamceller (MPP) som CD34 + CD38-CD90dimCD45RA-celler og CD34 + CD38-CD90-CD45RA + lymfoide-pulverlak Multipotente stamceller (LMPP) celler kan bestemmes i fleste af AML tilfælde8 ,9. Gennemsnitlige fluorescens-intensiteten (MFI) af CD38 er især vigtigt at tages i betragtning inden for den CD34 + celle rum. Fordi CD38 intensitet definerer tre nye celle segmenter heraf: CD38 negativ (P6), CD38 dim (P7) og CD38 lyse (P8) (figur 1). MFI CD38 kan bestemmes ved hjælp af enten hematogones og/eller plasma celler som en indre positiv kontrol, da disse celler kraftigt express CD38.
Immundefekte SCID-NOD-IL2Rγcnull (NSG) musemodel er almindeligt anvendt til engraftment af normal, og ondartet menneskelige hæmatopoietisk celler10,11. Seriel xenotransplantation assays bruges til at validere eksperimentelt LSC eller HSC funktion. Disse undersøgelser er blevet grundigt analyseret af stor skala sekventering metoder. Ikke desto mindre er mindre kendt om LSC og HSC immunophenotype af AML blast befolkning aflægger i NSG mus i forhold til dens primære AML (figur 2).
Antallet af LSC er i sagens natur lav således, identifikation og kvantificering af LSC er udfordrende og metoden beskrevet her kan bruges som en flow cytometric assay på diagnose og kliniske følge at evaluere kemoterapi svar (som resterende LSC kan forårsage AML tilbagefald). Tilstedeværelsen af LSC kan indikere positiv minimal residual sygdom (MRD). Til dato, MRD overvågning i AML hovedsageligt er afhængige af molekylære metoder (dvs., RT-PCR, NGS)10,12. Men i denne protokol vi registrerer samtidige protein udtryk for flere HSC/LSC markører (CD34, CD38, CD45RA, CD90) på primære blast celler af human AML af multiparametric flow flowcytometri2,8,9. Denne kombination af antistoffer kan anvendes i enhver standard flow flowcytometri laboratorium og dette flow MRD assay er særlig interessant, når MRD af real-time Polymerasekædereaktionen (RT-PCR) ikke er muligt, dvs., hvis leukæmi specifikke molekylære markører er ikke kan påvises i den diagnostiske AML prøve. Denne protokol er desuden supplerer de mere følsomme molekylære MRD teknikker, som det har til formål at påvise og bestemme de unormale hæmatopoietisk stamceller som repræsenterer en funktionel celle karakteristik (figur 3).
Vi viser hvordan man kan kvantificere tre hæmatopoietisk stamfader befolkninger og den formodede LSC rum med forskellig grad af modning ved flowcytometri med antistoffer findes i fleste af kliniske laboratorier. Desuden vi bekræftet tilstedeværelsen af disse celle rum i tilsvarende patient-afledt-xenografts (PDX) og på behandling opfølgning.

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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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		<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>
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			<td height="20" style="height:21px;">PBS</td>
			<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 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>
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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>
			<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>
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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>
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		<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>
		</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>
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			<td height="20" style="height:21px;">Anti-CD3-PE</td>
			<td>Beckman Coulter</td>
			<td>IM1451</td>
			<td></td>
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		<tr height="20">
			<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>
		</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>
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		<tr height="20">
			<td height="20" style="height:21px;">Navios</td>
			<td>Beckman Coulter</td>
			<td>DS-14644A</td>
			<td style="width:467px;"></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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		<tr height="20">
			<td height="20" style="height:21px;">CST BD</td>
			<td>BD Biosciences</td>
			<td>655051</td>
			<td></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>
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		<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>
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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

Akut myeloid leukæmi (AML) er en heterogen, og hvis de ikke behandles, dødelig sygdom. Det er den mest almindelige årsag til leukæmi-associerede dødelighed hos voksne. I første omgang er AML en sygdom i hæmatopoietisk stamceller (HSC) karakteriseret ved anholdelsen af differentiering, efterfølgende ophobning af blast leukæmiceller og reduceret produktion af funktionelle hæmatopoietisk elementer. Heterogenitet strækker sig til tilstedeværelsen af leukæmi stamceller (LSC), med denne dynamiske celle rum udvikler sig for at overvinde forskellige udvalg pres, der er pålagt under leukæmi progression og behandling. For yderligere at definere LSC befolkningen, tillader tilsætning af CD90 og CD45RA forskelsbehandling af normale HSCs og Multipotente stamceller inden for CD34 + CD38-celle rum. Her, skitsere vi en protokol for at opdage samtidig udtryk for flere formodede LSC markører (CD34, CD38, CD45RA, CD90) på primære blast celler af human AML ved multiparametric flowcytometri. Derudover viser vi hvordan du kvantificere tre stamfader befolkninger og en formodede LSC befolkningen med stigende grad af modning. Vi bekræftet tilstedeværelsen af disse befolkningsgrupper i tilsvarende patient-afledt-xenografts. Denne metode til påvisning og kvantificering af formodede LSC kan anvendes til klinisk opfølgning af kemoterapi svar (dvs., minimal residual sygdom), som resterende LSC kan forårsage AML tilbagefald.

Her vil vi præsentere en metode til at bestemme stamfader populationer i normal, og ondartet menneskelige knoglemarv prøver. Vi har indsamlet knoglemarven og milt fra en vellykket PDX og udført multiparametric flowcytometri, som beskrevet ovenfor. Vi sammenlignede flere delpopulationer af blast celler mellem diagnostiske patient prøven og den tilsvarende PDX og især CD34 + CD38-stamfader rum, især beriget i formodede LSC. Vi har fundet, at CD34 + CD38-blast brøkdel var højere i PDX i forhold til den diagnostiske ...

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

Vi skitsere en protokol for at opdage samtidig udtryk for leukæmi stamceller markører på primære akut myeloid leukæmiceller ved flowcytometri. Vi viser hvordan man kan kvantificere tre stamfader befolkninger og en formodede LSC befolkningen med stigende grad af modning. Vi bekræftet tilstedeværelsen af disse befolkningsgrupper i tilsvarende patient-afledt-xenografts.

Flow flowcytometri for at vurdere leukæmi stamceller i primære akut Myeloid leukæmi og Patient-afledt-xenografts, på diagnose og følg op

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

Omfattende protokol til prøven og proces knoglemarven til at måle målbare Residual sygdom og Leukemic stamceller i akut Myeloid leukæmi

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

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

Forberedelse af primære akut lymfoblastær leukæmiceller i forskellige cellecyklus faser af Centrifugal Elutriation

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

Isolering og karakterisering af tumor initierende celler fra sarkom patient afledte Xenografts

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

En kromatin Immunoprecipitation analyse til at identificere nye NFAT2 Target gener i kronisk lymfatisk leukæmi

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

Vurdering af primære leukæmiceller metaboliske profil

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

Flow Cytometrisk analyse af mitokondrie reaktive oxygen arter i murine hæmatopoietiske stamceller og Progenitorcellerne og MLL-AF9 drevet leukæmi

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

Subcellulære fraktionering af primære kroniske Lymfocytiske leukæmiceller til at overvåge nuklear/cytoplasmisk protein handel

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

Test målrettede behandlinger i kræft ved hjælp af strukturel dna ændringsanalyse og patient-afledte Xenografts

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

To Flow cytometriske tilgange af NKG2D Ligand Surface Detection at skelne stamceller fra Bulk Subpopulations i akut myeloid leukæmi

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

Intra-peritoneal transplantation til generering af akut myeloid leukæmi hos mus

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