This research has been supported by Dutch Cancer Society (ALPE 2013-6371) and Egbers foundation for VONK. We thank the Dutch AML-MRD working group for the collaboration and fruitful discussions for continuous improvement and standardization of AML-MRD.

<ol>
	<li>D&#246;hner, H., 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=Diagnosis+and+management+of+AML+in+adults:+2017+ELN+recommendations+from+an+international+expert+panel.">Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel.</a> Blood. 129 (4), 424-447 (2017).</li><li>Zwaan, C. 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=Collaborative+Efforts+Driving+Progress+in+Pediatric+Acute+Myeloid+Leukemia.">Collaborative Efforts Driving Progress in Pediatric Acute Myeloid Leukemia.</a> J Clin Oncol. 33 (27), 2949-2962 (2015).</li><li>Cornelissen, J. 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=The+European+LeukemiaNet+AML+Working+Party+consensus+statement+on+allogeneic+HSCT+for+patients+with+AML+in+remission:+an+integrated-risk+adapted+approach.">The European LeukemiaNet AML Working Party consensus statement on allogeneic HSCT for patients with AML in remission: an integrated-risk adapted approach.</a> Nat Rev Clin Oncol. 9 (10), 579-590 (2012).</li><li>Grimwade, D., Freeman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+minimal+residual+disease+in+acute+myeloid+leukemia:+which+platforms+are+ready+for+&amp;#34;prime+time&amp;#34;?.">Defining minimal residual disease in acute myeloid leukemia: which platforms are ready for &amp;#34;prime time&amp;#34;?.</a> Hematology. 2014 (1), 222-233 (2014).</li><li>Kr&#246;nke, 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=Monitoring+of+minimal+residual+disease+in+NPM1-mutated+acute+myeloid+leukemia:+a+study+from+the+German-Austrian+acute+myeloid+leukemia+study+group.">Monitoring of minimal residual disease in NPM1-mutated acute myeloid leukemia: a study from the German-Austrian acute myeloid leukemia study group.</a> J Clin Oncol. 29 (19), 2709-2716 (2011).</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=High+prognostic+impact+of+flow+cytometric+minimal+residual+disease+detection+in+acute+myeloid+leukemia:+data+from+the+HOVON/SAKK+AML+42A+study.">High prognostic impact of flow cytometric minimal residual disease detection in acute myeloid leukemia: data from the HOVON/SAKK AML 42A study.</a> J Clin Oncol. 31 (31), 3889-3897 (2013).</li><li>van der Velden, V. H. 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=Clinical+significance+of+flowcytometric+minimal+residual+disease+detection+in+pediatric+acute+myeloid+leukemia+patients+treated+according+to+the+DCOG+ANLL97/MRC+AML12+protocol.">Clinical significance of flowcytometric minimal residual disease detection in pediatric acute myeloid leukemia patients treated according to the DCOG ANLL97/MRC AML12 protocol.</a> Leukemia. 24 (9), 1599-1606 (2010).</li><li>Ossenkoppele, G., Schuurhuis, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRD+in+AML:+time+for+redefinition+of+CR?.">MRD in AML: time for redefinition of CR?.</a> Blood. 121 (12), 2166-2168 (2013).</li><li>Feller, 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=MRD+parameters+using+immunophenotypic+detection+methods+are+highly+reliable+in+predicting+survival+in+acute+myeloid+leukaemia.">MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia.</a> Leukemia. 18 (8), 1380-1390 (2004).</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>Kalina, 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=EuroFlow+standardization+of+flow+cytometer+instrument+settings+and+immunophenotyping+protocols.">EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols.</a> Leukemia. 26 (9), 1986-2010 (2012).</li><li>Paietta, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+minimal+residual+disease+(MRD)+in+leukemia:+a+changing+definition+and+concept?.">Assessing minimal residual disease (MRD) in leukemia: a changing definition and concept?.</a> Bone Marrow Transplant. 29 (6), 459-465 (2002).</li><li>Loken, M. R., 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=Residual+disease+detected+by+multidimensional+flow+cytometry+signifies+high+relapse+risk+in+patients+with+de+novo+acute+myeloid+leukemia:+a+report+from+Children's+Oncology+Group.">Residual disease detected by multidimensional flow cytometry signifies high relapse risk in patients with de novo acute myeloid leukemia: a report from Children's Oncology Group.</a> Blood. 120 (8), 1581-1588 (2012).</li><li>Zeijlemaker, 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+simple+one-tube+assay+for+immunophenotypical+quantification+of+leukemic+stem+cells+in+acute+myeloid+leukemia.">A simple one-tube assay for immunophenotypical quantification of leukemic stem cells in acute myeloid leukemia.</a> Leukemia. 30 (2), 439-446 (2015).</li><li>. Guidance for Collection, Transport and Submission of Specimens for Ebola Virus Testing Available from: <a target="_blank" href="https://www.cdc.gov/vhf/ebola/healthcare-us/laboratories/specimens.html">https://www.cdc.gov/vhf/ebola/healthcare-us/laboratories/specimens.html</a> (2017)</li><li>Bachas, C., 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+role+of+minor+subpopulations+within+the+leukemic+blast+compartment+of+AML+patients+at+initial+diagnosis+in+the+development+of+relapse.">The role of minor subpopulations within the leukemic blast compartment of AML patients at initial diagnosis in the development of relapse.</a> Leukemia. 26 (6), 1313-1320 (2012).</li><li>Ravandi, 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=Persistence+of+minimal+residual+disease+assessed+by+multiparameter+flow+cytometry+is+highly+prognostic+in+younger+patients+with+acute+myeloid+leukemia.">Persistence of minimal residual disease assessed by multiparameter flow cytometry is highly prognostic in younger patients with acute myeloid leukemia.</a> Cancer. 123 (3), 426-435 (2017).</li><li>Hourigan, C. S., 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=Measurable+residual+disease+testing+in+acute+myeloid+leukaemia.">Measurable residual disease testing in acute myeloid leukaemia.</a> Leukemia. 31 (7), 1482-1490 (2017).</li></ol>

The antibody cocktails used for the validation of the stem cell tube in several independent institutes were kindly provided by BD. The authors have nothing to disclose in relation to this manuscript.

Ample data are available that show evidence for MRD positivity being associated with poor survival, and therefore MRD assessment may improve patient outcome by providing additional prognostic information upon which clinical decisions can be made. Hence, a consistent and harmonized method for immuno-phenotypic assessment of MRD is essential to ultimately improve patient therapy. This is also important when comparing clinical studies across different clinical sites, and may ultimately help in clinical decision making and serving as a surrogate clinical endpoint for overall survival. The notion that solid guidelines are warranted for accurate and consistent MRD measurements has led to a concerted action of the European Leukemia Network (ELN) to design state of the art guidelines. This comprehensive document will be published late-2017, and will be instrumental for many study groups and laboratories moving forward. 
Critical steps within the protocol
An important, and often overlooked aspect of MRD analysis is the impact of sample quality on accurate determination of MRD. This is more apparent when material has to be transported to other institutes in global clinical trials given the additional operational considerations that need to be taken into account in this setting. To reduce the risk of mixing up patient information and timely delivery of the samples adequate administration is crucial. Again, at this stage of the transport the correct forms and/or import into electronic hospital systems with clear assignments of the requested analysis is required. Noting the stage at therapy of MRD sampling is also crucial since it may be relevant for clinical decision making (for example after second course of therapy) and should therefore be defined clearly. The use of mock data (such as for instance January 1 per year of birth) increases the risk of mixing up patients or analyses. If required by law, an alternative anonymized way of identification should be used.
All specimens for immunophenotyping should be processed preferentially within 24 h of collection. Although not recommendable, bone marrow and peripheral blood samples can still be processed and analyzed when kept up to 72 h at ambient temperature. In addition, all handlings with the material can best be performed under sterile conditions, so further cryopreservation of cells in (local) biobanks remains relevant: many clinical studies have additional analyses to further investigate leukemia cells with respect to molecular, immuno-phenotypic, and functional features to be performed at a later stage. However details of biobanking are not within the scope of this manuscript.
Using MRD as a diagnostic tool implies that it needs an accredited laboratory, not only for flow cytometry, but also regarding quality control of MRD assessment. This may require distributing samples to specific reference laboratories or (re)analyzing flow files with the MRD measurements by reference teams. 
This article describes the most important actions from bone marrow aspirate collection to determination of MRD in clinical samples - requiring an entire team of experts with specific tasks, responsibilities, and with frequent communication for effective characterization and analysis. Since each task is crucial to the procedure, it is recommended to have sound logistics including protocols at each laboratory and sufficient back-up personnel who have been specifically trained for the task. In addition, since there are some relatively subjective steps in part of the procedures (especially LAIP and LSC aberrant marker identification), it is essential to have discussions about the final result by the team, and have the final report authorized by a team of supervisors. Current efforts are pursuing computer software development that will help standardize the (LAIP and LSC) MRD analysis.
Modification and troubleshooting
Each lab can have its own set of antibodies that can be used to define the different subpopulations although having a standardized backbone of markers is essential for comparable results, as outlined in the ELN state of the art guidelines for MRD measurements document referred to above. Irrespective of the chosen markers there are some issues that can make the analysis of the results difficult and need to be taken into consideration. 
Limitations of the technique
The identification of LAIP and LSC aberrant marker expression is first assessed at diagnosis and monitored over time (during and after therapy) for accurate characterization of the MRD phenotype. While over 95% of the patients can be evaluated via LAIP or LSC (or both), still some patients have no defined LAIPs or no CD34+CD38- LSCs or present with CD34 negative blasts, or have a missing diagnosis sample. In these cases, it is still worthwhile to try and measure MRD with a panel of antibodies as broad as the number of available cells allows and then select the most reliable (giving the strongest distinction of leukemic cells) LAIP. Considering that a proportion of patients who do ultimately relapse do not completely resemble the diagnosis immunophenotype, due to the heterogeneity of AML disease, measuring a broad panel of antibodies at MRD is recommended anyhow. These immunophenotypic shifts include blast cells and LSCs and have been shown to occur during therapy16 and the measureable disease may be based on these so-called upcoming populations. At this time however, it has not been determined whether all immunophenotypic sub-populations will lead to disease relapse, and is therefore not common practice to report MRD tailored-therapy based on these cells in clinical trials. It is important to note that the risk of missing LSC due to population shifts is reduced by the one tube approach in which the most important aberrancy defining markers are in one flow cytometry (PE) channel14.
Significance with respect to existing methods
The method described in this protocol relies on the definition of one or more LAIPs, which cells are followed during therapy. A disadvantage of this method is that it has been recently shown that LAIP may change during therapy. This way some upcoming populations with different aberrant markers may be missed. To circumvent this, it would be best to measure all aberrant markers instead of only those found at diagnosis. This would then be similar to the "different from normal" approach that is used by several laboratories17.
Future applications
Recently, MRD has received extra attention for novel clinical trial design. In the era of specialized treatment options and targeted drug therapy, the use of MRD as an outcome measure will reduce the time needed to establish clinical efficacy of novel treatments, ultimately allowing faster introduction of urgently needed therapeutic options into the clinic. The significance of appropriate logistics and practical execution of a harmonized MRD assessment are crucial to future AML treatment success as the FDA is currently investigating the feasibility of using MRD as a surrogate endpoint instead of overall survival measures18.

Acute myeloid leukemia (AML) is a malignancy of the bone marrow characterized by defects in the maturation program, with abnormal proliferation and accumulation of myeloid progenitor cells, inhibition of normal hematopoiesis, and ultimately bone marrow failure. The disease is highly heterogeneous with respect to morphology, immunophenotype, cytogenetics, molecular aberrations, and gene expression signatures, as well as to treatment response and treatment outcome1,2. Current management includes induction chemotherapy with the aim to achieve complete remission (CR), followed by post-remission treatment, which is largely guided by the results of molecular, cytogenetic and immunophenotypic studies and consists of either several courses of additional chemotherapy or (autologous or allogeneic) stem cell transplantation3. Despite high remission rates after intensive chemotherapy of up to 90%, 5-year survival in adults is only approximately 30%-40%, predominantly due to the development of relapses which are commonly resistant to chemotherapy and thereby very difficult to treat. Outcome in children is better, although approximately one third also relapse. Therefore, early detection of imminent relapse will fill an unmet medical need and may guide post-remission therapy4. 
Residual disease after therapy may reflect the sum of all diagnosis and post-diagnosis resistance mechanisms/factors; hence its measurement may be prognostic and instrumental for guiding treatment. The possibility of de&#64257;ning residual disease (formerly called minimal residual disease and now referred to as measurable residual disease or MRD) far below the morphological criterion of 5% blast cells is changing the landscape of risk classi&#64257;cation. Currently the two methods mostly used to detect MRD are flow cytometry-based and molecular-based, the latter being assessed by reverse transcriptase PCR (RT-qPCR)5 or, although in a premature stage, by next generation sequencing (NGS). Many studies in adults as well as in children already demonstrated that various MRD approaches provide strong prognostic information in AML both after induction and consolidation therapy6,7, and a new definition of disease burden (superior to morphological CR) is now emerging8. This suggests that MRD assessed by flow and/or molecular techniques should become, and in fact are already becoming, standard in every clinical trial in AML. 
This manuscript discusses the detailed flow cytometry procedure to obtain an accurate and reproducible immunophenotypic characterization of MRD in bone marrow samples, including the bone marrow sampling and processing procedures preceding flow cytometry. The availability of good quality bone marrow samples at diagnosis and follow-up is crucial to the success of this measurement across clinical sites and clinical trials. In fact, these pre-analytical considerations are also of vital importance for molecular (PCR and NGS) MRD approaches. For immunophenotypic characterization of MRD, aberrantly expressed markers are combined with normal myeloid and progenitor markers, to identify a leukemia-associated immunophenotype (LAIP)9. MRD measures the resultant of many factors influencing response to therapy, such as intrinsic or acquired leukemia drug resistance, pharmacodynamics and kinetics of therapy, immune surveillance, and compliance. Therefore, MRD is a very strong post-diagnosis prognostic parameter associated with clinical outcome when dichotomized on a cut-off level determined by Receiver-Operating Characteristic (ROC) analysis. For our adult AML cohort of the HOVON 42a study, the cut-off level is set at 0.1% of LAIP positive cells/total white blood cells. Using this criterion for determining negative vs positive MRD status, a group of patients can be identified who have a significantly worse relapse incidence, relapse-free and overall survival6. Additionally, we describe the measurement of immature, drug resistant leukemia cells with stem cell-like features (CD34+CD38- leukemia stem cells, or LSC), which offers a strong predictor of patient outcome10. Together LAIP and LSC approaches form the flow cytometric MRD approach. The LAIP approach is suitable for roughly 90% of the patients, while the LSC approach can be applied in about 80% of the patients. Together over 95% of the patients can be evaluated for either one parameter or both. 
Lastly, this publication provides a detailed operational description to assess MRD by flow cytometry. This includes: 1) harmonization and/or standardization of bone marrow sampling procedures, 2) sample transportation guidance 3) detailed description of the leukemic cell detection with FACS using several antibody panels including single cell tube approaches to characterize the LSC, 4) set-up of the FACS machines for standardized measurements, 5) analytical programs for MRD measurements and 6) analytical programs for LSC detection.
We aim to show all facets of the procedure including the sample preparation since that is rarely discussed while it is an important issue for the quality of the ultimate result. Bone marrow aspiration and biopsy are clinical procedures used to evaluate the hematopoietic cells within bone marrow. These are performed together with a complete blood count (CBC) and blood smear. The optimal method for bone marrow aspiration is crucial for the accurate diagnosis and follow-up for MRD measurement. In addition, a successful bone marrow aspirate should contain enough cells to perform the LAIP and LSC flow analysis (at least 10 million viable cells). Here, we describe the method for performing a bone marrow aspiration and provide guidelines, which should result in adequate cell sampling (and limit the potential for hemodilution) needed for accurate diagnostics and additional research. These pre-analytical considerations are also of vital importance for molecular (PCR and NGS) MRD approaches. All specimens for immunophenotyping should be processed preferentially within 24 h of collection. Although not recommendable, bone marrow and peripheral blood samples can still be processed and analyzed when kept up to 72 h at ambient temperature. In addition, all handlings with the material should be performed under sterile conditions, in order to enable cryopreservation of sterile cells for later research/quality assessment etc.

<table><tbody><tr><td></td><td>BD Biosciences</td><td>120995496,060996589 and 013729</td><td>FACS Canto II&amp;nbsp;</td></tr><tr><td></td><td>BD Biosciences</td><td>555360</td><td>CD7 FITC</td></tr><tr><td></td><td>DAKO</td><td>F076701</td><td>CD2 FITC</td></tr><tr><td></td><td>Sanquin</td><td>M1613</td><td>CD36 FITC</td></tr><tr><td></td><td>BD Biosciences</td><td>332778</td><td>CD15 FITC</td></tr><tr><td></td><td>BD Biosciences</td><td>335039</td><td>CD45RA FITC</td></tr><tr><td></td><td>BD Biosciences</td><td>345810</td><td>CD56 PE</td></tr><tr><td></td><td>BD Biosciences</td><td>337899</td><td>CD22 PE</td></tr><tr><td></td><td>BD Biosciences</td><td>345785</td><td>CD14 PE</td></tr><tr><td></td><td>Miltenyi Biotec</td><td>130-080-801</td><td>CD133 PE</td></tr><tr><td></td><td>BD Biosciences</td><td>562566</td><td>Clec12a PE</td></tr><tr><td></td><td>BD Biosciences</td><td>565568</td><td>Tim-3 PE</td></tr><tr><td></td><td>BD Biosciences</td><td>332774</td><td>CD7 PE</td></tr><tr><td></td><td>BD Biosciences</td><td>333142</td><td>CD11b PE</td></tr><tr><td></td><td>BD Biosciences</td><td>337899</td><td>CD22 PE</td></tr><tr><td></td><td>BD Biosciences</td><td>345810</td><td>CD56 PE</td></tr><tr><td></td><td>BD Biosciences</td><td>347222</td><td>CD34 PERCP-CY5.5</td></tr><tr><td></td><td>BD Biosciences</td><td>558714</td><td>CD123 PERCP-CY5.5</td></tr><tr><td></td><td>Beckman Coulter</td><td>B49221</td><td>CD117 PE-CY7</td></tr><tr><td></td><td>BD Biosciences</td><td>562854</td><td>CD33 BV421</td></tr><tr><td></td><td>BD Biosciences</td><td>345791</td><td>CD19 APC</td></tr><tr><td></td><td>BD Biosciences</td><td>333143</td><td>CD11b APC</td></tr><tr><td></td><td>BD Biosciences</td><td>345800</td><td>CD33 APC</td></tr><tr><td></td><td>BD Biosciences</td><td>345807</td><td>CD38 APC</td></tr><tr><td></td><td>BD Biosciences</td><td>641411</td><td>HLA-DR APC-H7</td></tr><tr><td></td><td>BD Biosciences</td><td>560532</td><td>CD44 APC-H7</td></tr><tr><td></td><td>BD Biosciences</td><td>562596</td><td>CD13 BV421</td></tr><tr><td></td><td>BD Biosciences</td><td>348811</td><td>CD34 PE-CY7</td></tr><tr><td></td><td>Beckman Coulter</td><td>A96416</td><td>CD45 Krome Orange</td></tr><tr><td></td><td>BD Biosciences</td><td>655873</td><td>CD45 HV500c</td></tr><tr><td></td><td>BD Biosciences</td><td>655051</td><td>CST beads&amp;nbsp;</td></tr><tr><td></td><td>BD Biosciences</td><td>555899</td><td>Pharm lysing solution</td></tr><tr><td></td><td>BD Biosciences</td><td>644204</td><td>Multicolor CompBeads</td></tr><tr><td></td><td>BD Biosciences</td><td>655051</td><td>CST beads</td></tr><tr><td></td><td>BD Biosciences</td><td></td><td>FACSDiva software</td></tr><tr><td></td><td>Cytognos</td><td></td><td>Infinicyt&amp;nbsp;</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>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>HSA</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>Azide</td></tr><tr><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;</td><td>ddH2O</td></tr></tbody></table>

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

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

In 90% of AML patients at least one LAIP can be identified. In order to generate the accurate percentages of LAIP+ cells of the primitive blast cells at diagnosis, the appropriate setting of the blast gate is crucial and needs verification as visualized in Figure 3. An example of each patient harboring a specific type of aberrancy on the CD34+ primitive cells is visualized in Figure 5. For measurement of MRD at follow-up,the earlier defined LAIP+ cells are gated and defined as percentage of LAIP+ cells of the WBC. Hence the setting of this gate is crucial to achieve the right MRD results. Figure 6 shows a patient who remains in complete remission and a patient that relapses after having MRD positive results (&#8805; 0.1 %) after the second cycle of induction therapy.
The current protocol is only suitable to define stem cells in CD34+ cells, since thorough characterization of this compartment has shown that the CD38- population harbors the most potent stem cell like fraction. In order to separate the LSC from the HSC within this fraction, additional markers are used. Most often selected markers to distinguish LSC are CD45RA and the combi channel harboring 6 specific LSC markers labeled with PE. Based on further clinical evaluations, a cut-off level of &#8805; 0.004% was defined as LSC-positive and was associated with worse survival at diagnosis while a cut-off level of 0.0001% was used at follw-up10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56386/56386fig1.jpg" /> 
Figure 1: Bone marrow smear. A) healthy donor and B) AML patient (FAB 5 subtype). Giemsa stain shown at 100x magnification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56386/56386fig2.jpg" /> 
Figure 2: Correct packaging material for biological fluids. More details on the rules and regulations of transporting biological fluids can be found at the website15 of Centers for Disease Control and Prevention. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56386/56386fig3.jpg" /> 
Figure 3: Gating strategy for defining blast cells. A) Gating CD45dim, B) Gating CD34+ blasts.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56386/56386fig4.jpg" /> 
Figure 4: Gating lymphocytes. A) Blue cells are WBC shown in FSC/SSC plot, B) Green cells are lymphocytes characterized by CD45high/SSClow, C) Examples of negativity for CD34, D) negativity for CD117 and E) negativity for CD13, F) The different cell types are shown in an overview population tree. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56386/56386fig5.jpg" /> 
Figure 5: LAIPs at AML diagnosis. A) LAIP based on cross lineage aberrancy (CD7 on CD33 expressing cells), B) LAIP based on asynchronous differentiation (CD11b on CD13 expressing cells), C) LAIP based on overexpression compared to normal cells (CD34very high on CD13 expressing cells), D) LAIP based on underexpression compared to normal cells (HLA-DRlow on CD33 expressing cells). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56386/56386fig6.jpg" /> 
Figure 6: LAIP followed during therapy. A) LAIP definition at diagnosis (CD34+/CD13+/CD33-) and loss of LAIP during follow-up in a patient who remained in continuous complete remission, B) LAIP definition at diagnosis (CD117+/CD7+/CD33+) and persistence of LAIP during follow-up in a patient who relapsed. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56386/56386fig7.jpg" /> 
Figure 7: Stem cell gating. A) Gating of the different CD34+/CD38 populations based on beads (CD38low is below the upper border of the beads, while CD38very low are defined as the true negative cells representing the LSC and are located below the median of the beads), B) two examples of patients with distinction between HSC and LSC at follow-up based on CD45RAneg and CD45RApos expression. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/56386fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/Sampling_Form_MRD_1.pdf" target="_blank">Please click here to downoad this file.</a>
Supplemental File 2. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/Sampling_Form_MRD_2.pdf" target="_blank">Please click here to downoad this file.</a>
Supplemental File 3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56386/Supplementary_information_revised_Cloos_et_al.zip" target="_blank">Please click here to downoad this file.</a>

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

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

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

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

29.1K Views.  VU University Medical Center. Detectie van minimale of meetbare residuele ziekte (MRD) is een belangrijke voorspellende biomarker voor risico-evaluatie te verfijnen en het voorspellen van herval in acute myeloïde leukemie (AML). Deze uitgebreide richtlijnen en aanbevelingen met de best practices voor consistente en accurate identificatie en opsporing van MRD, kan steun bij het maken van effectieve AML behandelingsbeslissingen.

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

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

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

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

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

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

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

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

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

Assessment of the Metabolic Profile of Primary Leukemia Cells

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

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

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

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

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

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

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell disease. This observation has tremendous implications, as subsequent therapeutic immunomodulation strategies can improve clinical outcome. Conventionally, polymerase chain reaction-based analysis of short tandem repeats is used to identify chimerism in donor-derived blood cells. However, this method is restricted to nucleated cells and cannot distinguish between dissociated single-cell lineages. We applied the analysis of short tandem repeats to flow cytometric-sorted hematopoietic progenitor cells and compared this with the analysis of short tandem repeats obtained from selected burst-forming unit - erythroid colonies, both collected from the bone marrow. With this method we are able to demonstrate the different proliferation and differentiation of donor cells in the erythroid compartment. This technique is eligible to complete current monitoring of chimerism in the stem cell transplant setting and thus may be applied in future clinical studies, stem cell research and design of gene therapy trials.

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

The presence of incomplete chimerism is noted in a large proportion of patients following bone marrow transplant for thalassemia major or sickle cell ...

Developmental Biology

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Medicine

Intrafemoral Injection for Bone Marrow Study in Live Mice

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Leukemic Subpopulation Harvest: A Method for Spatial Separation of Leukemic Cell Subpopulations from 2D Co-culture

Source: Slone, W. L., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/53645">Modeling Chemotherapy Resistant Leukemia In Vitro.</a> J. Vis. Exp. (2016).
This video describes a method for spatial separation of three leukemic cell subpopulations: the suspended leukemic cells freely floating in the media, phase bright leukemic cells adhered to the surface of the mesenchymal stromal cell monolayer, and the phase dim leukemic cells that have migrated beneath the mesenchymal stromal cell monolayer from 2D co-culture.

Source: Slone, W. L., et al. Modeling Chemotherapy Resistant Leukemia In Vitro. J. Vis. Exp. (2016).
This video describes a method for spatial separation ...

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

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