scholarly journals Semicontinuous flow centrifugation for the pheresis of immunocompetent cells and stem cells

Blood ◽  
1977 ◽  
Vol 49 (3) ◽  
pp. 391-397 ◽  
Author(s):  
RS Weiner ◽  
CM Richman ◽  
RA Yankee
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Buffy Coat ◽  
Immunocompetent Cells ◽  
Antitumor Therapy ◽  
Volume Analysis ◽  
Large Numbers

Mononuclear cells from human peripheral blood were purified by semicontinuous flow centrifugation (SCFC) using the Haemonetics model 30 blood cell separator; 64% +/- 7% of the mononuclear cells in 600 ml of peripheral blood were collected in a 30-ml volume. Analysis of sequential 5-ml aliquots of the mononuclear cell concentrate revealed that both immunocompetent cells and granulocytic progenitor cells (CFU- C) were proportional to the cell count throughout the buffy coat. In vitro pheresis of large volumes of human bone marrow resulted in recovery of 63% of the cells, 12% of the hemoglobin, and 84% of the CFU- C in 20% of the original volume. Further centrifugation eliminated 80% of the platelets without loss of cells or CFU-C. SCFC of peripheral blood or bone marrow selectively concentrated mononuclear cells and reduced the contamination by granulocytes and erythrocytes. Large numbers of mononuclear cells can thus be collected for studies in vitro or for cryopreservation and the autologous reconstitution of immunosuppressed or myelosuppressed patients undergoing intensive antitumor therapy.

scholarly journals Semicontinuous flow centrifugation for the pheresis of immunocompetent cells and stem cells

Blood ◽  
1977 ◽  
Vol 49 (3) ◽  
pp. 391-397 ◽  
Author(s):  
RS Weiner ◽  
CM Richman ◽  
RA Yankee
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Buffy Coat ◽  
Immunocompetent Cells ◽  
Antitumor Therapy ◽  
Volume Analysis ◽  
Large Numbers

Abstract Mononuclear cells from human peripheral blood were purified by semicontinuous flow centrifugation (SCFC) using the Haemonetics model 30 blood cell separator; 64% +/- 7% of the mononuclear cells in 600 ml of peripheral blood were collected in a 30-ml volume. Analysis of sequential 5-ml aliquots of the mononuclear cell concentrate revealed that both immunocompetent cells and granulocytic progenitor cells (CFU- C) were proportional to the cell count throughout the buffy coat. In vitro pheresis of large volumes of human bone marrow resulted in recovery of 63% of the cells, 12% of the hemoglobin, and 84% of the CFU- C in 20% of the original volume. Further centrifugation eliminated 80% of the platelets without loss of cells or CFU-C. SCFC of peripheral blood or bone marrow selectively concentrated mononuclear cells and reduced the contamination by granulocytes and erythrocytes. Large numbers of mononuclear cells can thus be collected for studies in vitro or for cryopreservation and the autologous reconstitution of immunosuppressed or myelosuppressed patients undergoing intensive antitumor therapy.

scholarly journals Increase in circulating stem cells following chemotherapy in man

Blood ◽  
1976 ◽  
Vol 47 (6) ◽  
pp. 1031-1039 ◽  
Author(s):  
CM Richman ◽  
RS Weiner ◽  
RA Yankee
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cancer Patients ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Intensive Chemotherapy ◽  
Large Numbers ◽  
Intermittent Chemotherapy

Abstract The number of circulating granulocytic stem cells (CFU-C) was determined by the in vitro methylcellulose technique in cancer patients receiving intermittent chemotherapy. In 17 patients studied prior to therapy, the median CFU-C concentration per 2 X 10(5) mononuclear cells plated was six, compared to a posttreatment median of 23 in 21 patients (p less than 0.001). Large numbers of stem cells were obtained by leukopheresis and cryopreserved with a 99.5% median CFU-C recovery. Cyclical changes in the concentration of stem cells with maximum values of 20 times baseline were demonstrated in a patient studied at weekly intervals during multiple courses of treatment. It was estimated that, at peak CFU-C concentrations, a quantity of stem cells equivalent to that present in a bulk bone marrow harvest could be obtained from the peripheral blood by a 17-liter pheresis. These results suggest that it may be practical to obtain an adequate number of stem cells from the peripheral blood to study autologous stem cell infusion as a means of averting myelosuppression in patients receiving intensive chemotherapy.

scholarly journals Increase in circulating stem cells following chemotherapy in man

Blood ◽  
1976 ◽  
Vol 47 (6) ◽  
pp. 1031-1039 ◽  
Author(s):  
CM Richman ◽  
RS Weiner ◽  
RA Yankee
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cancer Patients ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Intensive Chemotherapy ◽  
Large Numbers ◽  
Intermittent Chemotherapy

The number of circulating granulocytic stem cells (CFU-C) was determined by the in vitro methylcellulose technique in cancer patients receiving intermittent chemotherapy. In 17 patients studied prior to therapy, the median CFU-C concentration per 2 X 10(5) mononuclear cells plated was six, compared to a posttreatment median of 23 in 21 patients (p less than 0.001). Large numbers of stem cells were obtained by leukopheresis and cryopreserved with a 99.5% median CFU-C recovery. Cyclical changes in the concentration of stem cells with maximum values of 20 times baseline were demonstrated in a patient studied at weekly intervals during multiple courses of treatment. It was estimated that, at peak CFU-C concentrations, a quantity of stem cells equivalent to that present in a bulk bone marrow harvest could be obtained from the peripheral blood by a 17-liter pheresis. These results suggest that it may be practical to obtain an adequate number of stem cells from the peripheral blood to study autologous stem cell infusion as a means of averting myelosuppression in patients receiving intensive chemotherapy.

Leukoreduction System Chambers as a Source of Viable Human Peripheral Blood Mononuclear Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4912-4912
Author(s):  
Sinyoung Kim ◽  
Han-Soo Kim ◽  
Yangsoon Lee ◽  
Jaewoo Song ◽  
Hyun Ok Kim ◽  
...  
Keyword(s):  
Cell Count ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Buffy Coat ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
The Mean

Abstract Many blood banks are using apheresis machines to collect blood components such as platelets (PLTs), RBCs, or plasma. Especially, leukoreduced plateletpheresis using apheresis instrument (Trima Accel, Gambro BCT, Lakewood, CO) provided subsidiary cell products retained in leukoreduction system (LRS) chamber that was originally discarded. The LRS chamber is a conical-shaped chamber that uses saturated, fluidized, particle bed filtration technology to remove WBCs from PLTs. In the current study, a total of 24 LRS chambers from different donors were investigated to determine it would be a valuable source of viable human peripheral blood mononuclear cells (MNCs). The proportions of CD3+, CD19+, CD16+/CD56+, CD14+, CD45+ cells, and absolute CD34+ cell count within the LRS chambers were determined by flow cytometry. Dendritic cells (DCs) were generated from the immunomagnetically purified CD14+ cells from LRS chamber and characterized by phenotypic surface marker and stimulatory capacity in an allogeneic mixed lymphocyte reaction. In the LRS chamber, the total number of WBC count was 1.1 × 109 ± 0.3 × 109 and the mean percentage of MNCs was 80.6 ± 13.1%. The mean proportion of T cells, B cells, NK cells, CD14+ monocytes among CD45+ cells was 54.3 ± 11.5%, 6.4 ± 3.1%, 14.6 ± 3.9%, 12.9 ± 7.5%, respectively. Total absolute CD34+ cell count in LRS chamber was 0.95 × 106 ± 0.65 × 106. Also, we could demonstrate CD14+ cells isolated from LRS chamber was capable of differentiating into functionally mature DCs in vitro. LRS chambers are a valuable and convenient source of viable human peripheral blood mononuclear cell population and could replace standard buffy coat preparations for research applications.

scholarly journals Proangiogenic Cell Colonies Grown In Vitro from Human Peripheral Blood Mononuclear Cells

2012 ◽  
Vol 17 (9) ◽  
pp. 1128-1135 ◽  
Author(s):  
Kreton Mavromatis ◽  
Diane J. Sutcliffe ◽  
Giji Joseph ◽  
R. Wayne Alexander ◽  
Edmund K. Waller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Antigen Expression ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Surface Antigen Expression

Although multiple culture assays have been designed to identify endothelial progenitor cells (EPCs), the phenotype of cells grown in culture often remains undefined. We sought to define and characterize the proangiogenic cell population within human peripheral blood mononuclear cells. Mononuclear cells were isolated from peripheral blood and grown under angiogenic conditions for 7 days. Formed colonies (CFU-As) were identified and analyzed for proliferation, mRNA and surface antigen expression, tube-forming ability, and chromosomal content. Colonies were composed of a heterogeneous group of cells expressing the leukocyte antigens CD45, CD14, and CD3, as well as the endothelial proteins vascular endothelial (VE) cadherin, von Willebrand’s factor (vWF), CD31, and endothelial nitric oxide synthase (eNOS). Colony cells expressed increased levels of proangiogenic growth factors, and they formed tubes in Matrigel. In comparison with colonies from the CFU-Hill assay, our assay resulted in a greater number of colonies (19 ± 9 vs. 13 ± 7; p < 0.0001) with a substantial number of cells expressing an endothelial phenotype (20.2% ± 7.4% vs. 2.2% ± 1.2% expressing eNOS, p = 0.0006). Chromosomal analysis indicated the colony cells were bone marrow derived. We, therefore, describe a colony-forming unit assay that measures bone marrow–derived circulating mononuclear cells with the capacity to proliferate and mature into proangiogenic leukocytic and endothelial-like cells. This assay, therefore, reflects circulating, bone marrow–derived proangiogenic activity.

scholarly journals Binding of fluorescein-labeled anaphylatoxin C5a to human peripheral blood, spleen, and bone marrow leukocytes

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 152-160
Author(s):  
T Werfel ◽  
M Oppermann ◽  
M Schulze ◽  
G Krieger ◽  
M Weber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Human Peripheral Blood ◽  
Myeloid Cells ◽  
Lymphocyte Subpopulation ◽  
Marrow Cells

The expression of C5a receptors (C5aR) on human leukocytes was evaluated by flow cytometry using fluorescein-labeled human C5a (C5a- F). Granulocytes and CD14+ mononuclear cells (MNL) but not CD3+, CD20+, CD16+, CD56+, or CD11b+ lymphocytes in peripheral blood and spleen bound C5a-F. C5a-F binding was saturable and inhibitable by anti-C5a monoclonal antibody (MoAb) C17/5 or unlabeled C5a. During hemodialysis, which led to the generation of C5a, only granulocytes and monocytes increased their expression of the adhesion molecule CD11b (CR3). In vitro, C5a induced an increase of CR3 and p 150/95 (CD11c/CR4) only on myeloid cells. However, treatment of leukocytes with phorbol 12- myristate 13 acetate increased CR3 and CR4 expression on both myeloid cells and a lymphocyte subpopulation. Stimulation of MNL in mixed lymphocyte cultures or by treatment with conditioned medium or with IFN- gamma did not induce binding sites for C5aR on lymphocytes and reduced the binding of C5a-F to monocytes. The expression of C5aR on low- density bone marrow cells was analyzed by setting appropriate gates during flow cytometry. Cells that bound C5a-F were found in all populations that contained granulocyte and monocyte precursors, but not in lymphocyte precursor populations. All C5aR+ bone marrow cells were CD34 and expressed high levels of CR3, which suggests a late appearance of C5aR during myeloid cell maturation. Our results indicate that C5aR is exclusively expressed on myeloid cells within the hematopoetic cell population.

scholarly journals Binding of fluorescein-labeled anaphylatoxin C5a to human peripheral blood, spleen, and bone marrow leukocytes

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 152-160 ◽  
Author(s):  
T Werfel ◽  
M Oppermann ◽  
M Schulze ◽  
G Krieger ◽  
M Weber ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Human Peripheral Blood ◽  
Myeloid Cells ◽  
Lymphocyte Subpopulation ◽  
Marrow Cells

Abstract The expression of C5a receptors (C5aR) on human leukocytes was evaluated by flow cytometry using fluorescein-labeled human C5a (C5a- F). Granulocytes and CD14+ mononuclear cells (MNL) but not CD3+, CD20+, CD16+, CD56+, or CD11b+ lymphocytes in peripheral blood and spleen bound C5a-F. C5a-F binding was saturable and inhibitable by anti-C5a monoclonal antibody (MoAb) C17/5 or unlabeled C5a. During hemodialysis, which led to the generation of C5a, only granulocytes and monocytes increased their expression of the adhesion molecule CD11b (CR3). In vitro, C5a induced an increase of CR3 and p 150/95 (CD11c/CR4) only on myeloid cells. However, treatment of leukocytes with phorbol 12- myristate 13 acetate increased CR3 and CR4 expression on both myeloid cells and a lymphocyte subpopulation. Stimulation of MNL in mixed lymphocyte cultures or by treatment with conditioned medium or with IFN- gamma did not induce binding sites for C5aR on lymphocytes and reduced the binding of C5a-F to monocytes. The expression of C5aR on low- density bone marrow cells was analyzed by setting appropriate gates during flow cytometry. Cells that bound C5a-F were found in all populations that contained granulocyte and monocyte precursors, but not in lymphocyte precursor populations. All C5aR+ bone marrow cells were CD34 and expressed high levels of CR3, which suggests a late appearance of C5aR during myeloid cell maturation. Our results indicate that C5aR is exclusively expressed on myeloid cells within the hematopoetic cell population.

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