Critical role of flow cytometry in evaluating peripheral blood hematopoietic stem cell grafts

Keeney, Michael; Gratama, Jan W.; Sutherland, D. Robert

doi:10.1002/cyto.a.10103

Cited by 24 publications

(16 citation statements)

References 30 publications

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“…Despite these differences, clinical hematology has been greatly facilitated by advances in flow cytometry. The distributed nature of the hematopoietic system makes it readily amenable to flow cytometric analysis and many surface proteins and glycoproteins on stem cells, erythrocytes, leukocytes, and platelets have been studied in great detail [12,13]. Moreover, many leukemias can be identified by abnormal CD45 expression and side scatter properties with co-expression of other molecules used to discriminate the phenotype, for example CD34 + CD13 + for acute myeloid leukemias [14].…”

Section: Resultsmentioning

confidence: 99%

Mouse hematopoietic stem cell identification and analysis

et al. 2008

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Hematopoietic stem cells (HSCs) remain by far the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains difficult because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in bone marrow is about 0.01% of total nucleated cells and $5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. This prohibits the study of processes in HSCs, which require large amounts of starting material. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. This report will review different hematopoietic stem and progenitor purification strategies and compare flow cytometry profiles for HSC sorting and analysis on different instruments. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. The purpose of this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology. ' International Society for Advancement of CytometryKey terms hematopoietic stem cell; flow cytometry; peripheral blood; cell cycle HEMATOPOIETIC stem cells have tremendous therapeutic potential and have been harnessed in the clinic for more than 40 years in the context of bone marrow transplantation. Multipotent long-term HSCs (LT-HSCs) reside in the bone marrow and through a process of asymmetric cell division, can self-renew to sustain the stem cell pool or differentiate into short-term HSCs (ST-HSCs) or lineage-restricted progenitors that undergo extensive proliferation and differentiation to produce terminally differentiated, functional hematopoietic cells. ST-HSCs or multipotent progenitors (MPPs) are only able to sustain hematopoiesis in the short term, whereas the LTHSCs must persist for the lifespan of the organism to perpetually replenish the hematopoietic system. HSCs can be isolated from bone marrow or peripheral blood using enrichment (magnetic cell separation-MACS) and/or single-cell sorting (fluorescence-activated cell sorting-FACS) based on cell surface markers and/or vital dye staining. The HSC has served as the paradigm for adult stem cell populations by virtue of a well-defined differentiation cascade with distinct intermediaries connecting the differentiation of LT-HSCs into mature, functional hematopoietic cells. Each of the cell stages of HSC differentiation can be purified from the bone marrow or peripheral blood using characteristic cell surface markers, which has greatly facilitated the study of hemato...

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Section: Resultsmentioning

confidence: 99%

Mouse hematopoietic stem cell identification and analysis

et al. 2008

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“…Multicolor flow cytometry (MFC) is widely used in health research and treatment for a variety of tasks, such as providing the counts of helper-T lymphocytes needed to monitor the course and treatment of human immunodeficiency virus (HIV) infection [1-3], diagnosing and monitoring leukemia and lymphoma patients [4, 5], and evaluating peripheral blood hematopoietic stem cell grafts [6] and a variety of other diseases [7]. The technology is also used to cross-match organs for transplantation [8], and in research involving stem cells, apoptosis [9], phagocytosis [10], and a wide range of cellular properties including phenotype [11], cytokine expression [12], and cell-cycle status [13].…”

Section: Introductionmentioning

confidence: 99%

Single-color Multitarget Flow Cytometry Using Monoclonal Antibodies Labeled with Different Intensities of the Same Fluorochrome

Park

Han

2012

Ann Lab Med

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BackgroundWe developed a single-color multitarget flow cytometry (SM-FC) assay, a single-tube assay with graded mean fluorescence intensities (MFIs). We evaluated the repeatability of SM-FC, and its correlation with multicolor flow cytometry (MFC), to assess its application as a routine FC assay.MethodsWe selected CD19, CD3, CD4, and CD8 as antigen targets to analyze a lymphocyte subset. MFIs were graded by adjusting monoclonal antibody (mAb) volumes to detect several cell populations. Dimly labeled mAb was prepared by decreasing mAb volume and the optimum diluted volume was determined by serial dilution. SM-FC repeatability was analyzed 10 times in 2 normal controls. The correlation between SM-FC and MFC was evaluated in 20 normal and 23 patient samples.ResultsCV values (0.8-5.0% and 1.3-4.1% in samples 1 and 2, respectively) acquired by SM-FC with CD3-fluorescein α-isothyocyanate (FITC)dim+CD4-FITCbright and with CD19-FITCdim+CD3-FITCbright showed good repeatability, comparable to that acquired by MFC (1.6-3.7% and 1.0-4.8% in samples 1 and 2, respectively). Excellent correlation was observed between the 2 methods in the 20 normal samples (B cells, T cells, non-Thelper cells, and Thelper cells; r2=0.87, 0.97, 0.97, and 0.98, respectively; P<0.05). There were also linear relationships between SM-FC with CD19-FITCdim+CD3-FITCbright and CD8-PEdim+CD4-PEbright, and MFC, in the 23 patient samples (B cells, T cells, Tcytotoxic cells, and Thelper cells; r2≥0.98, 0.99, 0.99, and 0.99, respectively; P<0.05).ConclusionsThe multicolor, single-tube SM-FC technique is a potential alternative tool for identifying a lymphocyte subset.

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“…Flow cytometry (FCM) is widely used in health research and treatment for a variety of tasks, such as providing the counts of helper-T lymphocytes needed to monitor the course and treatment of HIV infection, in the diagnosis and monitoring of leukemia and lymphoma patients, the evaluation of peripheral blood hematopoietic stem cell grafts, and many other diseases [1–8]. The technology is also used in cross-matching organs for transplantation, research involving stem cells, vaccine development, apoptosis, phagocytosis, and a wide range of cellular properties including phenotype, cytokine expression, and cell-cycle status [9–14].…”

Section: Introductionmentioning

confidence: 99%

A Survey of Flow Cytometry Data Analysis Methods

Bashashati

Brinkman

2009

Advances in Bioinformatics

112

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Flow cytometry (FCM) is widely used in health research and in treatment for a variety of tasks, such as in the diagnosis and monitoring of leukemia and lymphoma patients, providing the counts of helper-T lymphocytes needed to monitor the course and treatment of HIV infection, the evaluation of peripheral blood hematopoietic stem cell grafts, and many other diseases. In practice, FCM data analysis is performed manually, a process that requires an inordinate amount of time and is error-prone, nonreproducible, nonstandardized, and not open for re-evaluation, making it the most limiting aspect of this technology. This paper reviews state-of-the-art FCM data analysis approaches using a framework introduced to report each of the components in a data analysis pipeline. Current challenges and possible future directions in developing fully automated FCM data analysis tools are also outlined.

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Critical role of flow cytometry in evaluating peripheral blood hematopoietic stem cell grafts

Cited by 24 publications

References 30 publications

Mouse hematopoietic stem cell identification and analysis

Mouse hematopoietic stem cell identification and analysis

Single-color Multitarget Flow Cytometry Using Monoclonal Antibodies Labeled with Different Intensities of the Same Fluorochrome

A Survey of Flow Cytometry Data Analysis Methods

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