The antibodies used in this study were provided by BD Biosciences. The authors would like to thank their colleague, Dr. Pascale Flandrin-Gresta, from the Department of Molecular Biology, Hematology laboratory, University Hospital of Saint-Etienne, France, who provided expertise for interpretation of NGS data for the second MDS case. The authors are thankful for the clinician hematologists for their interest and involvement in this study and for the patients and healthy donors for their agreement to participate in this study. The authors would also like to thank the "Les Amis de R&#233;mi" Foundation for financial support for publication.

<ol><li>Orfao, A., Ortuño, F., de Santiago, M., Lopez, A., San Miguel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunophenotyping+of+Acute+Leukemias+and+Myelodysplastic+Syndromes%5BTitle%5D)+AND+%22Cytometry+Part+A%22%5BJournal%5D)">Immunophenotyping of Acute Leukemias and Myelodysplastic Syndromes.</a> Cytometry Part A. 58, 62-71 (2004).</li>
<li>Porwit, A., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/24919805">Revisiting guidelines for integration of flow cytometry results in the WHO classification of Myelodysplastic Syndromes - proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS (IMDSFlow).</a> Leukemia. 28 (9), 1793-1798 (2014).</li>
<li>Westers, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implementation+of+flow+cytometry+in+the+diagnostic+work-up+of+myelodysplastic+syndromes+in+a+multicenter+approach%3A+Report+from+the+Dutch+Working+Party+on+Flow+Cytometry+in+MDS%5BTitle%5D)+AND+%22Leukemia+Research%22%5BJournal%5D)">Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach: Report from the Dutch Working Party on Flow Cytometry in MDS.</a> Leukemia Research. 36, 422-430 (2012).</li>
<li>Meehan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AutoGate%3A+automating+analysis+of+flow+cytometry+data%5BTitle%5D)+AND+%22Immunologic+Research%22%5BJournal%5D)">AutoGate: automating analysis of flow cytometry data.</a> Immunologic Research. 58, 218-223 (2014).</li>
<li>Greenberg, P. L., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/21233243">NCCN Clinical Practice Guidelines in Oncology: myelodysplastic syndromes.</a> Journal of the National Comprehensive Cancer Network. 9, 30-56 (2011).</li>
<li>Greenberg, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myelodysplastic+Syndromes%2C+Version+2.2017%2C+Clinical+Practice+Guidelines+in+Oncology%5BTitle%5D)+AND+%22Journal+of+the+National+Comprehensive+Cancer+Network%22%5BJournal%5D)">Myelodysplastic Syndromes, Version 2.2017, Clinical Practice Guidelines in Oncology.</a> Journal of the National Comprehensive Cancer Network. 15, (2017).</li>
<li><a target="_blank" href="https://www.amazon.com/Classification-Tumours-Haematopoietic-Lymphoid-Tissues/dp/928324494X">WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th Edition</a>. Swerdlow, S. H., et al. (2), IARC Press. Lyon, France. 86-128 (2017).</li>
<li>https://lists.purdue.edu/pipermail/cytometry/2009-March/036889.html. , Available from: 
 <a target="_blank" href="https://lists.purdue.edu/pipermail/cytometry/2009-March/036889.html">https://lists.purdue.edu/pipermail/cytometry/2009-March/036889.html</a> (2018).</li>
<li><a target="_blank" href="NULL">Composition of EuroFlow panels and technical information on reagents Version 1.5</a>. , (2017).</li>
<li><a target="_blank" href="NULL">EuroFlow Standard Operating Procedure (SOP) for sample preparation and staining Version 1.2.1</a>. , (2015).</li>
<li>Kalina, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EuroFlow+standardization+of+flow+cytometer+instrument+settings+and+immunophenotyping+protocols%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols.</a> Leukemia. 26, 1986-2010 (2012).</li>
<li>http;//www.miltenyibiotec.com/en/products-and-services/macs-flow-cytometry/reagents/antibodies-and-dyes/recombinant-antibodies.aspx. , Available from: 
 <a target="_blank" href="http;//www.miltenyibiotec.com/en/products-and-services/macs-flow-cytometry/reagents/antibodies-and-dyes/recombinant-antibodies.aspx">http;//www.miltenyibiotec.com/en/products-and-services/macs-flow-cytometry/reagents/antibodies-and-dyes/recombinant-antibodies.aspx</a> (2018).</li>
<li>van Dongen, J. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EuroFlow+antibody+panels+for+standardized+n-dimensional+flow+cytometric+immunophenotyping+of+normal%2C+reactive+and+malignant+leukocytes%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes.</a> Leukemia. 26, 1908-1975 (2012).</li>
<li>Matarraz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introduction+to+the+Diagnosis+and+Classification+of+Monocytic-Lineage+Leukemias+by+Flow+Cytometry%5BTitle%5D)+AND+%22Cytometry+Part+B+%28Clinical+Cytometry%29%22%5BJournal%5D)">Introduction to the Diagnosis and Classification of Monocytic-Lineage Leukemias by Flow Cytometry.</a> Cytometry Part B (Clinical Cytometry). 92 (3), 218-227 (2015).</li>
<li>Westers, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunophenotypic+analysis+of+erythroid+dysplasia+in+myelodysplastic+syndromes.+A+report+from+the+IMDSFlow+working+group%29%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A report from the IMDSFlow working group).</a> Haematologica. 102, 308-319 (2017).</li>
<li>Shen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+immunophenotypic+findings+in+chronic+myelomonocytic+leukemia+and+its+utility+in+monitoring+treatment+response%5BTitle%5D)+AND+%22European+Journal+of+Haematology%22%5BJournal%5D)">Flow cytometry immunophenotypic findings in chronic myelomonocytic leukemia and its utility in monitoring treatment response.</a> European Journal of Haematology. 95, 168-176 (2015).</li>
</ol>

The authors declare that they have no competing financial interests. The Flow-Cytometry Department, Hematology Laboratory at the University Hospital of Saint-Etienne&#160;is a member of EuroFlow Consortium.

The quality of BM aspirate could impact on the final results. The hemodilution of the BM aspirate could distort the distribution of cells in different stages of maturation due to the absence of progenitors or precursors cells. Probably employing a bulk lysing method may help in normalization of BM aspirates for hemodilution in flow cytometric analyses. In addition, the critical steps for the evaluation of BM myeloid dysplasia by flow-cytometry are the sample processing and staining, data acquisition, and interpretation<s...

In the absence of phenotypic markers specific to the dysplastic changes occurring in myeloid cells during MDS initiation and progression, a new approach has been proposed in recent years based on the evaluation of the maturation pathways (altered expression of myeloid antigens during the production of mature myeloid cells) or of the abnormal distribution of different cell types within bone marrow (BM) cell compartments1,2.
This article presents a new method for standardized application of MFC in order to detect dysplastic changes in BM myeloid cell compartments related to myelodysplastic syndromes (MDS) or other myeloid hematological diseases. This study also shows the utility of using maturation databases for MFC data analysis.
Standardization of the sample preparation procedure, data acquisition, and analysis using the databases would allow the identification of the most relevant phenotypic abnormalities related to dysplastic changes in BM myeloid cells. Therefore, statistically selected subsets based on well-labeled and well-recognized formats (Automatic Population Separator (APS) diagrams, histograms, and dot plots) are required for developing an analysis strategy that can be used in subsequent analysis rounds. The discovery of robust phenotypic abnormalities in MDS would ease the diagnosis in cases with or without minimal morphological dysplasia and without cytogenetic aberrancies. Identification of discriminatory parameters allowing for the reduction of immunophenotypic panels may simplify the current scores2, permitting their applicability in clinical pathology&#160;laboratories.
This method limits the subjective interpretations of cytometry data, as have been signaled in MDS diagnosis3. This step is a prerequisite for the development of automated tools for processing and analyzing flow data4.
MDS comprises a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders in which the spliceosome mutations cooperate with specific epigenetic modifiers to yield the MDS phenotype. It is now known that, along with HSC mutations, other mechanisms are involved in MDS pathophysiology, such as aberrant immune-mediated inflammation and interactions between malignant HSCs and the stromal microenvironment of the BM. However, these mechanisms remain poorly understood. The wide clinical and biological heterogeneity of MDS makes the diagnosis and selection of the optimal therapy a challenge. In the last decade, multiple studies have shown that MFC is often more sensitive in detecting dysplasia2 than morphology, but technical and economic constraints make this technique difficult to standardize, with results often depending on the experience of the interpreter3. In addition, it is unclear how MFC can tip the balance toward MDS in cases with or without minimal morphological dysplasia and in the absence of cytogenetic anomalies, or in borderline cases such as hypocellular MDS, with a low blast count, from other non-clonal BM disorders such as bone marrow failure (i.e., aplastic anemia). It also remains difficult to differentiate borderline cases of MDS with an excess of blasts from acute myeloid leukemia (AML). For all these reasons, the clinical guidelines do not integrate MFC testing into the MDS final diagnosis. In 2011, the US National Comprehensive Cancer Network (NCCN) recommended MFC for the estimation of the percentage of CD34+ cells, detection of paroxysmal nocturnal hemoglobinuria clones, and presence of cytotoxic T-cell clones in hypocellular MDS5. These two latter situations also involve a therapeutic goal because clinical data have shown a good response of these patients to immunosuppressive therapy6. The 2017 NCCN guidelines, citing the International Working Group (IWG) recommendations, listed aberrant immunophenotyping detection by MFC among the co-criteria for MDS diagnosis, but without making any specifications6. In addition, the recently published WHO classification stipulates that MFC findings alone are not sufficient to establish a primary diagnosis of MDS in the absence of conclusive morphological and/or cytogenetic data7. However, MFC can be used as an additional test showing the dysregulation of myeloid cell maturation patterns and quantifying the &#34;distance from normal&#34; for a patient at a specific time in the disease course.
This method is applicable at clinical laboratories interested in the evaluation of dysplasia in BM myeloid cells using MFC immunophenotyping, in order to refine the diagnosis in MDS or other myeloid disorders with dysplastic abnormalities.

<table><tbody><tr><td>BD FACSCanto II flow-cytometer</td><td>BD Biosciences, CA, USA</td><td>SN: V33896301336</td><td>3-laser, 4-2-2 configuration, Filters and mirrors details: https://www.bdbiosciences.com/documents/BD_FACSCanto_II_FilterGuide.pdf</td></tr><tr><td>Awel C48-R Centrifuge</td><td>AWEL Industries, FR</td><td>SN: 910120016; Model No: 320002001</td><td>low speed centrifuges; capacity 60 FACS tubes</td></tr><tr><td>Pipetts of 10&amp;micro;L and 200&amp;micro;L</td><td></td><td></td><td></td></tr><tr><td>Pasteur pipettes</td><td></td><td></td><td></td></tr><tr><td>15 mL Falcon tubes</td><td></td><td></td><td></td></tr><tr><td>polypropylene tube for FACS</td><td></td><td></td><td></td></tr><tr><td>Mouse Anti-Human HLA-DR</td><td>BD Biosciences, CA, USA</td><td>655874</td><td>clone L243&lt;br/&gt; Mouse BALB/c IgG2a, &amp;kappa;&lt;br/&gt; Fluorochrome Horizon V450&lt;br/&gt; (Ex max 404 nm/&lt;br/&gt; Em max 448 nm)</td></tr><tr><td>Mouse Anti-Human CD45</td><td>BD Biosciences, CA, USA</td><td>560777</td><td>clone HI30&lt;br/&gt; Mouse IgG1, &amp;kappa;&lt;br/&gt; Fluorochrome Horizon V500 (Ex max 415 nm/&lt;br/&gt; Em max 500 nm)</td></tr><tr><td>Mouse Anti-Human CD16</td><td>BD Biosciences, CA, USA</td><td>656146</td><td>clone CLB/fcGran1 Mouse BALB/c IgG2a, &amp;kappa;&lt;br/&gt; Fluorochrome FITC&lt;br/&gt; (Ex max 494 nm/&lt;br/&gt; Em max 520 nm)</td></tr><tr><td>Mouse Anti-Human CD13</td><td>BD Biosciences, CA, USA</td><td>347406</td><td>clone L138&lt;br/&gt; Mouse BALB/c X C57BL/6 IgG1, &amp;kappa;&lt;br/&gt; Fluorochrome PE&lt;br/&gt; (Ex max 496 nm/&lt;br/&gt; Em max 578 nm)</td></tr><tr><td>Mouse Anti-Human CD34</td><td>BD Biosciences, CA, USA</td><td>347222</td><td>clone 8G12&lt;br/&gt; Mouse BALB/c IgG1, &amp;kappa;&lt;br/&gt; Fluorochrome PerCP-Cy5.5&lt;br/&gt; (Ex max 482 nm/&lt;br/&gt; Em max 678 nm)</td></tr><tr><td>Mouse Anti-Human CD117</td><td>BD Biosciences, CA, USA</td><td>339217</td><td>clone 104D2&lt;br/&gt; Mouse BALB/c IgG1&lt;br/&gt; Fluorochrome PE-Cy7&lt;br/&gt; (Ex max 496 nm/&lt;br/&gt; Em max 785 nm)</td></tr><tr><td>Mouse Anti-Human CD11b</td><td>BD Biosciences, CA, USA</td><td>333143</td><td>clone D12&lt;br/&gt; Mouse BALB/c IgG2a, &amp;kappa;&lt;br/&gt; D12, Fluorochrome APC&lt;br/&gt; (Ex max 650 nm/&lt;br/&gt; Em max 660nm</td></tr><tr><td>Mouse Anti-Human CD10</td><td>BD Biosciences, CA, USA</td><td>646783</td><td>clone HI10A&lt;br/&gt; Mouse BALB/c IgG1, &amp;kappa;&lt;br/&gt; Fluorochrome APC-H7&lt;br/&gt; (Ex max 496 nm/&lt;br/&gt; Em max 785nm)</td></tr><tr><td>Mouse Anti-Human CD35</td><td>BD Biosciences, CA, USA</td><td>555452</td><td>clone E11&lt;br/&gt; Mouse IgG1, &amp;kappa;&lt;br/&gt; Fluorochrome FITC&lt;br/&gt; (Ex max 494 nm/&lt;br/&gt; Em max 520 nm)</td></tr><tr><td>Mouse Anti-Human CD64</td><td>BD Biosciences, CA, USA</td><td>644385</td><td>clone 10.1&lt;br/&gt; Mouse BALB/c IgG1, &amp;kappa;&lt;br/&gt; Fluorochrome PE&lt;br/&gt; (Ex max 496 nm/&lt;br/&gt; Em max 578 nm)</td></tr><tr><td>Mouse Anti-Human CD300e</td><td>Immunostep</td><td>IREM2A-T100</td><td>clone UP-H2&lt;br/&gt; Mouse BALB/c IgG1, k&lt;br/&gt; Fluorochrome APC&lt;br/&gt; (Ex max 496 nm/&lt;br/&gt; Em max 578 nm)</td></tr><tr><td>Mouse Anti-Human CD14</td><td>BD Biosciences, CA, USA</td><td>641394</td><td>clone MoP9&lt;br/&gt; Mouse BALB/c IgG2b, &amp;kappa;&lt;br/&gt; Fluorochrome APC-H7&lt;br/&gt; (Ex max 496 nm/&lt;br/&gt; Em max 785nm)</td></tr><tr><td>Mouse Anti-Human CD36</td><td>BD Biosciences, CA, USA</td><td>656151</td><td>clone CLB-IVC7&lt;br/&gt; Mouse IgG1, &amp;kappa;&lt;br/&gt; Fluorochrome FITC&lt;br/&gt; (Ex max 494 nm/&lt;br/&gt; Em max 520 nm)</td></tr><tr><td>Mouse Anti-Human CD105</td><td>BD Biosciences, CA, USA</td><td>560839</td><td>clone 266&lt;br/&gt; Mouse BALB/c IgG1, &amp;kappa;&lt;br/&gt; Fluorochrome PE&lt;br/&gt; (Ex max 496 nm/&lt;br/&gt; Em max 578 nm)</td></tr><tr><td>Mouse Anti-Human CD33</td><td></td><td>345800</td><td>clone P67.6&lt;br/&gt; Mouse BALB/c IgG1, &amp;kappa;&lt;br/&gt; Fluorochrome APC&lt;br/&gt; (Ex max 496 nm/&lt;br/&gt; Em max 578 nm)</td></tr><tr><td>Mouse Anti-Human CD71</td><td>BD Biosciences, CA, USA</td><td>655408</td><td>clone M-A712&lt;br/&gt; Mouse BALB/c IgG2a, &amp;kappa;&lt;br/&gt; Fluorochrome APC-H7&lt;br/&gt; (Ex max 496 nm/&lt;br/&gt; Em max 785nm)</td></tr><tr><td>Lysing Solution 10x Concentrate (IVD)</td><td>BD Biosciences, CA, USA</td><td>349202</td><td></td></tr><tr><td>FACSFlow Sheath Fluid</td><td>BD Biosciences, CA, USA</td><td>342003</td><td></td></tr><tr><td>FACSDiva CS&amp;amp;T IVD beads</td><td>BD Biosciences, CA, USA</td><td>656046</td><td></td></tr><tr><td>RAINBOW CALIBRATION PARTICLES, 8 PEAKS</td><td>Cytognos, Salamanca, Spain</td><td>SPH-RCP-30-5A</td><td>lots EAB01, EAC01, EAD05, EAE01, EAF01, EAG01, EAH01, EAI01, EAJ01, EAK01</td></tr><tr><td>Compensation Particles Multicolor CompBeads(CE/IVD)</td><td>BD Biosciences, CA, USA</td><td>ref. #51-90-9001229 + #51-90-9001291</td><td></td></tr><tr><td>Diva software versions 6.1.2 and 6.1.3</td><td>BD Biosciences, CA, USA</td><td></td><td></td></tr><tr><td>Phosphate buffered saline tablets</td><td>R&amp;amp;D Systems, Minneapolis, USA</td><td>5564</td><td></td></tr><tr><td>Bovine serum albumin&amp;nbsp;(BSA)</td><td>Sigma-Aldrich, France</td><td>A9647</td><td></td></tr><tr><td>Sodium azide 99%</td><td>Sigma-Aldrich, France</td><td>199931</td><td></td></tr><tr><td>Infinicyt software version 1.8.0.e</td><td>Cytognos, Salamanca, Spain</td><td></td><td></td></tr></tbody></table>

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 54 BM samples harvested in K-EDTA anticoagulant were included in the study. The MFC data were analyzed in the absence of any information about the patients. Retrospective study showed that the BM samples were from 7 healthy donors (5 males and 2 females with a median age of 47.4 [35-48], 11 individuals with no evidence of a hematopoietic disease (8 males and 3 females with a median age of 57.9 [35-72]) and 36 cases with various pathological conditions: 1 case with anemia and low creat...

Watch this Scientific Journal Video about Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation at JoVE.com

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

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 ...

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Medicine

11.5K Views.  University Hospital of Saint-Etienne. Deze methode kan helpen om belangrijke vragen te beantwoorden bij de diagnose van myeloïde ziekten zoals myelodysplasiesyndromen of andere hematologische ziekten die evolueren met rijpingsafwijkingen in myeloïde celstamen. Het belangrijkste voordeel van deze techniek is dat het de subjectiviteit van de onderzoeker in die analyse en interpretatie vermindert. Deze methode kan inzicht geven in welke de meest discriminerende parameters voor myelodysplasie zijn.Het maakt kwantificering va...

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

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Immunology and Infection

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.

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 ...

Cancer Research

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 ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Developmental Biology

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 ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Biology

Flow Cytometry Analysis of Murine Bone Marrow Hematopoietic Stem and Progenitor Cells and Stromal Niche Cells

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As such, it contains hematopoietic stem and progenitor cells (HSPCs), as well as supporting stromal cells that contribute to the maintenance and regulation of HSPCs. Hematological and other BM disorders disrupt hematopoiesis by affecting hematopoietic cells directly and/or through the alteration of the BM niche. Here, we describe a method to study hematopoiesis in health and malignancy through the phenotypic analysis of murine BM HSPCs and stromal niche populations by flow cytometry. Our method details the required steps to enrich BM cells in endosteal and central BM fractions, as well as the appropriate gating strategies to identify the two key niche cell types involved in HSPC regulation, endothelial cells and mesenchymal stem cells. The phenotypic analysis proposed here may be combined with mouse mutants, disease models, and functional assays to characterize the HSPC compartment and its niche.

Here, we describe a simple protocol for the isolation and staining of murine bone marrow cells to phenotype hemopoietic stem and progenitor cells along with the supporting niche endothelial and mesenchymal stem cells. A method to enrich cells located in endosteal and central bone marrow areas is also included.

The bone marrow (BM) is the soft tissue found within bones where hematopoiesis, the process by which new blood cells are generated, primarily occurs. As ...

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

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