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At least 145 records · Page 8Linked to original sources
PubMed2007

Apheresis products of the Amicus and the AS.TEC 204 cell separators are comparable with regard to dendritic cells derived from the mononuclear cell collection.

BACKGROUND: In this study, we investigated the quality of autologous mononuclear cells (MNC) collected with two different cell separators using standard MNC-apheresis procedure modalities. MNCs were purified by density gradient centrifugation and cultured according to standard protocols to generate dendritic cells (DC) and 1 x 10(7)/ml immature DCs were pulsed with tumour lysate for 3 days and subsequently characterized by fluorescent-activated cell sorter analysis. RESULTS: No difference was found in the monocyte content of either apheresis product (P = 0.07) and in the overall yield of MNCs (P = 0.7). Mature DCs as defined by their phenotype revealed also no significant difference: Amicus, 118 x 10(6) cells +/- 91 vs. AS.TEC 204, 128 x 10(6) cells +/- 137 (P = 0.55), respectively, although the contamination with platelets (threefold) and red cells (twofold) was significantly higher in the AS.TEC 204 group (P < 0.05) than in the Amicus group. CONCLUSION: The Amicus and the AS.TEC 204 are equally capable in providing MNCs for the generation of DCs and the amount of concomitantly collected red cells and platelets had no impact on the final DC yield.

Adult↗
PubMedDate not supplied

[Progress in the therapy of children with congenital hemolytic anemias by transfusion of neocyte concentrations. Initial experiences in the separation and transfusion of young erythrocytes, obtained with the cell separator Fenwal CS-3000].

Transfusion-induced hemochromatosis remains a major therapeutic complication in the management of patients with chronic hemolytic anemias. Red cell units harvested with blood cell separators have an average estimated mean cell age of 30 days, compared to 60 days for unfractionated blood. Our initial experience with transfusion of neocytes indicates that the amount of transfused erythrocytes can be reduced on 60 per cent. Further studies must demonstrate the clinical benefit. When combined with modern iron chelator regimens, it may be possible to achieve consistently negative iron balance prior to the onset of hemochromatosis which would allow patients to probably lead a normal life-span.

Anemia, Hemolytic, Congenital↗
PubMed2006

CD 14+ cell collection in non-cytokine-stimulated donors with the COM.TEC cell separator.

BACKGROUND: The COM.TEC cell separator (Fresenius Hemocare), equipped with the MNC program (single-stage chamber) and the PBSC-LYM program (dual-stage chamber), was evaluated for CD 14+ cell collection regarding cell yields, collection efficiencies (CEs), and the content of residual cells in harvests. STUDY DESIGN AND METHODS: Twenty-four non-cytokine-stimulated donors underwent 5-L mononuclear cell (MNC) collections on the COM.TEC device to compare both programs. Two software versions (v02.03.05 vs. v 03.00.04) were investigated for optimization of the CD 14+ cell collection process. Blood counts of donors and products were analyzed for CD 14+ cells by flow cytometry and for platelets (PLTs), white blood cells, and red blood cells (RBCs) by a blood cell counter. RESULTS: In 5-L collections, the MNC program resulted in high CEs (83+/- 23%) and yields (1.2 x 10(9)+/- 0.6 x 10(9) per unit) of CD 14+ cells, but the products showed high residual PLTs. The use of a dual-stage chamber in the PBSC-LYM program produced a low content of residual PLTs (0.7 x 10(11) +/- 0.3 x 10(11) per unit) and RBCs but failed to reach a target of 1 x 10(9) CD 14+ cells. Modulated light to stabilize the buffy-coat detection by the interface monitor significantly improved CD 14+ cell enrichment. By use of the PBSC-LYM program, higher centrifuge velocity (1700 rpm [382 x g] vs. 1500 rpm [297 x g]) improved significantly CD 14+ cell yields (0.7 x 10(9) vs. 0.5 x 10(9) cells). CONCLUSION: A pure CD 14+ cell product with low numbers of residual cells was obtained by the PBSC-LYM program, which could be useful for good manufacturing practice-conformed production within closed systems. The MNC program offers the collection of high CD 14+ cell yields with excellent CEs but also high residual PLT counts.

Blood Donors↗
PubMed1977

Ultrastructural integrity of mouse testicular cells separated by velocity sedimentation.

Mouse testicular cells were examined ultrastructurally to determine whether the cells are damaged during the preparation of single-cell suspensions or during cell separation. The testicular cells were dissociated from seminiferous tubules by trypsinization and were fixed immediatley; fixed after being held in suspension for 4 h at 4 degrees C; or fixed after being separated into enriched fractions by sedimentation velocity either at unit gravity or by centrifugal elutriation. In general, the ultrastructural integrity of the cells, compared with that of corresponding testicular cells fixed in situ, was maintained during the dissociation and separation procedures. Ultrastructural abnormalities were most frequently produced in Sertoli cells and were occasionally observed in the acrosomes and nuclei of round spermatids. The cytoplasmic matrix of the midpiece of mature elongated spermatids or spermatozoa and the acrosomes of these cells were often disrupted. It is suggested that the dissociation procedures were responsible for most of the observed alterations of ultrastructural integrity.

Acrosome↗
PubMed1995

Comparison of different protocols in plateletpheresis with the haemonetics MCS 3p blood cell separator with respect to parameters of product quality.

BACKGROUND: In thrombocytapheresis, intermittent flow cell separators produce platelet concentrates (PC) with a higher leucocyte contamination than continuous flow blood cell separators. The new discontinuous flow cell separator Haemonetics MCS 3p offers a low-leucocyte solution for PC. The quality of PC obtained by the MCS 3p was investigated in this study. DESIGN: Prospective study. SETTING: Haemapheresis Unit of a University Clinic. PATIENTS: Healthy blood donors from the haemapheresis unit. MATERIALS AND METHODS: Platelet (PLT) yield, separation efficiency and white blood cell (WBC) contamination were studied in three different protocols. Two protocols used a haemocalculator, which calculated the target volume based on the donor's physical characteristics and the desired PLT yield for the procedure. Protocol I used 3,000 ml as target process volume and protocol II 3.3 x 10(11) as desired PLT yield. Protocol III was used without haemocalculator. Glucose, lactate, lactate dehydrogenase (LDH), morphology score and pH value were analysed to investigate the quality of the PC. RESULTS: Platelet yield and separation efficiency were not statistically different in the three protocols. Leucocyte contamination was lowest in the protocol without haemocalculator (median: 3.15 x 10(6), range 0.4-20.8 x 10(6)). Glucose, lactate, LDH and pH were not statistically different in the three protocols. Morphology score was best in protocol III. CONCLUSIONS: PLT collection with the MCS 3p blood cell separator results in sufficient thrombocyte yields. Using the haemocalculator we were not able to achieve the desired platelet yield. For this reason, and because of the higher WBC contamination in protocol II we prefer PLT collection without the haemocalculator. The quality of the platelet concentrates was good with respect to the parameters glucose, lactate, LDH, morphology score and pH.

Adult↗
PubMed1998

Cell separation based on the reversible interaction between calmodulin and a calmodulin-binding peptide.

A cell separation system based on the calcium-dependent interaction of calmodulin (CM) with a calmodulin-binding peptide (CBP) has been developed. The prototype of this system utilizes an indirect method to label the target cell population. Cells are first labeled with a primary monoclonal antibody directed to a specific cell surface antigen, then with a secondary affinity reagent, consisting of a polyclonal goat anti-mouse IgG (GAM-IgG) that has been cross-linked to a CBP derived from the sequence of the rabbit skeletal muscle myosin light chain kinase. In the presence of Ca2+, the CBP on the cells labeled with GAM-IgG-CBP binds to biotinylated calmodulin (CM-Biotin) with high affinity. The target cells are then captured with a solid-phase streptavidin. The unbound non-target cells are washed away and the immobilized target cells are released by chelating Ca2+ with EGTA. The specificity of the GAM-IgG-CBP and CM-Biotin and the feasibility of using this system to separate cells was demonstrated using the KG-1 human acute myelogenous leukemia cell line. KG-1 cells were fractionated on the basis of cell surface expression of HLA-DR. The cell selection reagents and the cell separation process did not affect KG-1 cell viability while cells selected by this procedure were 90% pure with a yield of 75%. This cell separation system also was used for rare cell isolation from normal human peripheral blood mononuclear cells. T cells expressing the Vbeta5 T cell receptor, which represent < 5% of the unfractionated cells, were isolated with 89% viability, 72% purity, 80% yield, and retained the ability to respond to activation signals as measured by blast transformation. The results from this study show that a cell selection system based on the reversible interaction between CM and a CBP can be applied to gently and efficiently isolate cells from a heterogeneous starting population that are free of the solid matrix without exposure to the stresses of mechanical or enzymatic release.

Biotin↗
PubMed2000

Apheresis-induced platelet activation:comparison of three types of cell separators.

BACKGROUND: Platelet-harvesting technology differs in various cell separators. Alteration in shear stress and biocompatibility of surfaces may give variable platelet activation and thereby affect the quality of the component. STUDY DESIGN AND METHODS: Four groups (n = 10) of single-needle apheresis procedures using three cell separators, were compared: 1) Spectra LRS, 90-minute harvesting time; 2) MCS+, 90-minute harvest; 3) Amicus, 90-minute; and 4) Amicus, 45-minute. Whole-blood samples were collected from the donors as were samples from the final components at intervals during the first 4 hours after cessation of the apheresis. Platelet activation status and platelet activation capacity after agonist stimulation were assessed by flow cytometry. RESULTS: No activated platelets were found in preapheresis and postapheresis samples from the donors. The platelets in the components from the Amicus (90-min) were significantly more activated than those in the other groups of components: that is, there was increased size of platelet aggregates, increased fraction of microparticles, increased degranulation, increased fibrinogen receptor activation, and decreased von Willebrand factor receptor expression. Moreover, the response of these platelets to agonist stimulation was reduced for all activation variables. CONCLUSIONS: After 90 minutes' processing time, platelets obtained with the Amicus cell separator were significantly more activated than platelets harvested with the Spectra and the MCS+.

Blood Component Removal↗
PubMed1997

Preparation of autologous bone marrow grafts for cryopreservation using the AS104 cell separator.

We evaluated the AS104 cell separator (Fresenius AG, Bad Homburg, Germany) for ex vivo processing of bone marrow (BM) grafts of 43 patients suffering from germ cell cancer (GCC, n = 22), acute lymphocytic leukemia (ALL, n = 13) and malignant lymphoma (ML, n = 8). Recoveries of total nucleated cells (TNC), mononuclear cells (MNC) and colony-forming units granulocyte-macrophage (CFU-GM) were determined in the BM concentrates prepared for cryopreservation. Hematopoietic reconstitution was analyzed in patients who underwent autologous transplantation following high-dose radio-/chemotherapy (HDRCT). Processing of the BM suspension with a median volume of 1,013 ml (range: 422-1,574) resulted in 156 ml (80-186) within 50-120 min (median: 90). In the BM concentrates, medians of 28.6% TNC (10.6-69.6), 37.9% MNC (22.3-86.4), and 52.4% CFU-GM (20.8-96.4) were recovered. Twenty-six patients underwent HDRCT with reinfusion of autologous BM and were evaluable for engraftment. They received a median of 0.8 x 10(8) MNC/kg (0.3-1.6 x 10(8)) and 2.2 x 10(4) CFU-GM/kg (0.6-12.8 x 10(4) for hematopoietic rescue. Engraftment with neutrophils > 500/microliter occurred in a median time of 12 days (8-33) in all patients. We conclude that ex vivo processing of autologous BM with median recovery rates of 37.9% for MNC, and 52.4% for CFU-GM, results in a cell population that can rescue patients from HDRCT. The described technique is convenient, time-efficient, and provides reliable results in preparing BM autografts for cryopreservation.

Adolescent↗
PubMed1987

Separation and functional analysis of bone marrow cells separated by rhodamine-123 fluorescence.

Mouse bone marrow (BM) cells were separated on the basis of fluorescence intensity after labeling with the supravital, fluorescing dye rhodamine 123 (Rh 123). Rh 123 accumulates in the mitochondria. The BM fractions were tested for the presence of spleen colony-forming units (CFU-S) that produced colonies at day 8 and day 12 after bone marrow transplantation; for the ability to rescue lethally irradiated mice; and for thymus-repopulating ability. The results showed that all the day-8 CFU-S incorporated a relatively large amount of Rh 123, while the day-12 CFU-S were stained heterogeneously. Survival after lethal irradiation, as expressed in the numbers of day-12 CFU-S transplanted, was predominately mediated by the weakly fluorescing fraction. However, early thymus repopulation, which is caused by prothymocytes contained in the graft, was mediated by the brightly fluorescing fraction. Data in the literature indicate that a high uptake of RH123 is correlated with cellular proliferation. This suggests that all the day-8 CFU-S and about 60% of the day-12 CFU-S are cycling. In contrast, less than 10% of both day-8 and day-12 CFU-S are killed by S-phase-specific agents. From this we conclude that a high uptake of Rh 123 depends on other factors in addition to the cell cycle status. It is suggested that differentiation processes in which the day-8 CFU-S appears to be involved also cause the presence of many or very active mitochondria. The difference between the weakly and brightly fluorescing fractions in the number of day-12 CFU-S required for 30-day survival after lethal irradiation, suggests that there is heterogeneity among the day-12 CFU-S population or that the fraction weakly labeled with Rh123 contains other, less mature, cells that are responsible for survival after lethal irradiation. Finally, the presence of prothymocytes in the brightly labeled fraction shows that these cells are different from the stem cells that protect lethally irradiated mice.

Animals↗
PubMed1990

Characterization of the efficiency of a cell separation process by the extent of elimination of a contaminating cell type.

A stepwise approach to the selection of an appropriate technique for a cell separation problem is presented in which the preparative purification of cells is linked to their analytical separation. We have introduced the extent of elimination of a contaminating cell type from the cell type which one chooses to purify, as a separation parameter that characterizes the efficiency of a separation process independently of the relative cell composition of the starting material. In order to compare different separation techniques, a preparative fraction boundary needs to be chosen between the cell types. We defined this boundary in terms of the physical property on which the separation is based such that yield and purity of the isolated cell suspension are optimized simultaneously. With this analytical approach, it was found that a similar elutriation technique separated human and equine mononuclear cells equally well and that the separability of human monocytes and lymphocytes improved when the cells were separated by increasing the limiting sedimentation coefficient value of the elutriation chamber in small increments.

Animals↗
PubMed2001

Human UC-blood banking: impact of blood volume, cell separation and cryopreservation on leukocyte and CD34(+) cell recovery.

BACKGROUND: UC blood represents an increasingly useful source of hematopoietic stem cells for BMT, although, currently, low cell numbers generally limit its use to pediatric patients. We have determined parameters that influence the recovery of viable cells during processing and cryopreservation, in an effort to set guidelines for determining whether a sample will yield sufficient cells to be of use in the transplant setting. METHODS: UC blood was collected from donors from January 1996 to December 1999. Volume was reduced using Ficoll, followed by cryopreservation under liquid nitrogen. Total leukocyte and CD34(+)-cell counts were determined prior to processing and a subset of samples was also assessed post-processing and post-cryopreservation. RESULTS: Outcomes for 3816 samples were analyzed to determine the correlation between cell number, cell type, volume, and time between collection and processing. A positive relationship was observed between volume and cell number for both leukocytes and the CD34(+) cells. This correlation allowed us to determine the number of leukocytes and CD34(+) cells expected from a sample based on volume, and to set guidelines for determining the practicality of storing any given sample prior to processing and cryopreservation. DISCUSSION: Measuring blood volume gives a very useful indication of the total leukocyte and CD34(+) cell number. The majority (75%) of cord-blood samples contain sufficient leukocytes for a pediatric transplant, and the number of cells available can be determined prior to processing by measuring blood volume.

Antigens, CD34↗
PubMed1977

Bone marrow cell separation on Ficoll gradient.

Cell fraction from rat and murine bone marrow were obtained by sedimentation at unit gravity. A linear gradient of polymerized sucrose (Ficoll) was used and some properties of individual cell fractions were studied. The 3rd cell fraction containing the lymphoblasts and lymphocytes contained also stem cells.

Animals↗
PubMed1984

[Work on the Haemonetics 30 blood cell separator].

Our experience with cell separator led us to the conclusion that platelet concentration is rather high (6,94 X 10(11)/L), especially in the part of separated blood which contains significant percentage of erythrocytes. A considerable amount of leukocytes (1,6 X 10(10)/L) can be separated as well. It should be mentioned that leukocyte concentrations are significantly higher when Dexan is administered intravenously and Plasmasteril is used. Therapeutic plasmapheresis requires different approach which varies from the usual work with blood donors, since complications can be expected. Appropriate choice of plasma substituents should be made, as well as constant monitoring of electrocardiographic changes in patients.

Humans↗
PubMed1982

Enrichment of terminal deoxynucleotidyl transferase activity by cell separation.

Terminal deoxynucleotidyl transferase is a unique DNA polymerase that can carry out DNA synthesis on an initiator molecule in the absence of a template. The usefulness of this enzyme as a biological marker for following patients during treatment and remission has been suggested. The potential usefulness of this enzyme in predicting the onset of relapse before any morphological indications has been demonstrated in chronic myelogenous leukemia patients in blast phase of the disease. In order to be able to detect low levels of TdT activity especially during remission phase, we have used cell separation techniques which can enrich cell populations containing TdT activity. A number of cell separation techniques have been developed to separate different cell types. We have used the techniques of unit gravity sedimentation and free flow electrophoresis to achieve enrichment of TdT positive cell populations. Our results show that up to 20 fold enrichment of TdT activity in normal human bone marrow can be accomplished by using cell separation techniques. With the use of free flow electrophoresis, we have achieved enrichment of TdT positive cell populations from normal human bone marrow, cells from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia in blast phase of the disease. No TdT positive cells were detected in patients with acute myelogenous leukemia. These cell separation techniques should prove to be useful in early detection of relapse in patients in remission.

B-Lymphocytes↗
PubMed2001

The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence.

BACKGROUND: Several processes of plant development, such as abscission, pollen release, fruit dehiscence, and seed dispersal, require organs or tissues to physically disassociate or split open. Due to the immobility of plant cells, these processes occur through coordinated mechanisms of cell separation that are not found in animals. Arabidopsis produces dry dehiscent fruits (siliques) making it a convenient system for the genetic study of cell separation associated with dehiscence. RESULTS: We describe here a novel mutation in Arabidopsis called alcatraz (alc), which prevents dehiscence of fruit by specifically blocking the separation of the valve cells from the replum. The ALC gene is shown to encode a protein related to the myc/bHLH family of transcription factors and is expressed in the valve margins of the silique, which is the site of cell separation during dehiscence. Detailed studies using TEM indicates that ALC enables cell separation in Arabidopsis fruit dehiscence by promoting the differentiation of a strip of labile nonlignified cells sandwiched between layers of lignified cells. Transgenic plants expressing antisense or dominant-negative ALC are defective in silique dehiscence. CONCLUSIONS: Cell separation in fruit dehiscence requires a specialized cell layer which is nonlignified and capable of autolysis, specified by a myc/bHLH protein encoded by ALC. These findings may have relevance to other processes requiring cell separation, as well as for the practical design of crops with reduced seed losses.

Amino Acid Sequence↗
PubMed2004

GPI7 involved in glycosylphosphatidylinositol biosynthesis is essential for yeast cell separation.

GPI7 is involved in adding ethanolaminephosphate to the second mannose in the biosynthesis of glycosylphosphatidylinositol (GPI) in Saccharomyces cerevisiae. We isolated gpi7 mutants, which have defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature. WSC1, RHO2, ROM2, GFA1, and CDC5 genes were isolated as multicopy suppressors of gpi7-2 mutant. Multicopy suppressors could suppress the growth defect of gpi7 mutants but not the cell separation defect. Loss of function mutations of genes involved in the Cbk1p-Ace2p pathway, which activates the expression of daughter-specific genes for cell separation after cytokinesis, bypassed the temperature-sensitive growth defect of gpi7 mutants. Furthermore, deletion of EGT2, one of the genes controlled by Ace2p and encoding a GPI-anchored protein required for cell separation, ameliorated the temperature sensitivity of the gpi7 mutant. In this mutant, Egt2p was displaced from the septal region to the cell cortex, indicating that GPI7 plays an important role in cell separation via the GPI-based modification of daughter-specific proteins in S. cerevisiae.

Amino Acid Sequence↗
PubMed1997

Automated blood component collection with the MCS 3p cell separator: evaluation of three protocols for buffy coat-poor and white cell-reduced packed red cells and plasma.

BACKGROUND: Automated collection of blood components with a cell separator (MCS 3p, Haemonetics), was performed according to three protocols. STUDY DESIGN AND METHODS: The first protocol provided 2 units of fresh-frozen plasma (FFP); and one buffy coat-poor red cell (RBC) concentrate in additive solution. The second protocol included an additional in-line filtration of the RBC in a closed system after storage at 4 degrees C for 24 hours. In the third protocol, an additional platelet concentrate (PC) was recovered from the buffy coat. Cell counts and biochemical characterization of the RBCs (n = 20 each) were determined on Days 0, 1, 14, 28, and 49. RESULTS: The RBC volume was 336 +/- 9 mL (first protocol), 337 +/- 7 mL (second protocol) and 293 +/- 12 mL (third protocol) with a hematocrit of 59 +/- 2, 53 +/- 3, and 61 +/- 5, percent respectively. On Day 49, hemolysis was 0.24 +/- 0.1 percent (first protocol), 0.33 +/- 0.32 percent (second protocol), and 0.38 +/- 0.1 percent (third protocol). The filtered RBC concentrate met the international standards for white cell-reduced RBCs. Filtration resulted in a clinically irrelevant increase of hemolysis. The in vitro RBC values (lactate dehydrogenase, 2-hydroxybutyrate dehydrogenase, hemolysis, potassium, 2,3 DPG, ATP) were at least equal to those in RBCs collected by conventional whole-blood donation. There is a trend toward extended preservation of 2,3 DPG in RBCs collected by apheresis. Two units of FFP could be collected with each donation (first protocol: 420 +/- 55 mL, 5.4 +/- 7 WBCs/microL, 6.5 +/- 5 x 10(3) platelets/microL; second protocol: 440 +/- 33 mL, 3 +/- 5.2 WBCs/microL, 32 +/- 12 x 10(3) platelets/microL; third protocol: 398 +/- 32 mL, 5 +/- 12 WBCs/microL; 3.4 +/- 3.5 x 10(3) platelets/microL). PCs prepared from the buffy coat collected by the third protocol contained 90 +/- 30 x 10(9) platelets in 88 +/- 14 mL of plasma. In vitro test results in these PCs were superior to those in PCs collected by conventional whole-blood donation. The procedure was well tolerated by all donors. No adverse reactions appeared. CONCLUSION: Erythroplasmapheresis with the MCS 3p cell separator is a useful alternative to conventional whole-blood donation and separation.

Adenosine Triphosphate↗