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PubMed1993

Improvement of separation efficiency and concentrate purity of the Fresenius cell separator AS 104: results of a multicenter study. Haemapheresis Scientific Workshop Group of DGTI.

The aim of this multicenter study, initiated by the Haemapheresis Scientific Workshop Group of the DGTI was to evaluate separation protocols for the cell separator AS 104, marketed by Fresenius, using modified software and parameters which were believed to allow a more effective platelet collection with a significantly lower leukocyte contamination of the concentrates. Plateletpheresis data from 950 runs in ten hemapheresis centers, using virtually the same equipment, identical pheresis protocols, and cell counting methods were registered and statistically analyzed for each center and machine-related differences. Additionally, the counting methods of the centers were controlled by bi-weekly external cell count trials, and the plateletpheresis data were corrected using the results of these cell count trials, to obtain a comparison of the two versions of the protocol independent of the center effect. For protocol (or software version) 4.1, 610 runs were registered. The results of cell countings (chamber) are (given as means+/-standard deviations) 3.452x10 11+/-1.009x10 11 for the platelet yield (or thrombocyte yield), 9x10 6+/-23x10 8 for leukocyte contamination, and 17x10 6+/-70x10 6 for the erythrocyte contamination, and 53%+/-13.5% for the extraction efficiency, respectively. For software version 4.4 with 340 runs, the results are 3.642x10 11+/-0.974x10 11 for the platelet yield, 15x10 6+/-74x10 8 for leukocyte contamination, 20x10 6+/-44x10 6 for erythrocyte contamination, and 59%+/-12.4% for the extraction efficiency, respectively. For the leukocyte and erythrocyte contaminations, the means and standard deviations must be interpreted carefully since the statistical distribution showed a considerable skewing of the data. From the automatic counts, marginally smaller means were found. The data were corrected by the values from the ring study; and for these mathematically corrected data, statistical tests showed a significant improvement in the extraction efficiency from software version 4.1 to 4.4. At the same time, the leukocyte contamination was significantly lower with version 4.4.

Blood Component Removal↗
PubMed1998

Comparison of CD34+ bone marrow cells purified by immunomagnetic and immunoadsorption cell separation techniques.

We tested two positive selection techniques for separation of CD34+ cells from bone marrow and analyzed the yields of CD34+ cells, BFU-E, CFU-GM, CFU-MK and LTC-IC after selection and expansion. An immunoadsorption procedure (CellPro) and an immunomagnetic (Baxter) CD34+ cell separation method were employed to purify the same bone marrow samples from seven normal subjects. Mean yields of CFU-GM and CFU-MK and absolute numbers of LTC-ICs were not different in the two purified cell populations. In contrast, the mean recovery of BFU-E was significantly lower for the immunoadsorption (21 +/- 14%) than for the immunomagnetic technique (44 +/- 27%). After separation, CD34+ cells were evaluated in 10-day liquid cultures for their expansion capacity in terms of total cells and progenitors. The expansion capacity of progenitors such as CFU-GM, CFU-MK and especially BFU-E selected by immunoadsorption was higher than the capacity of progenitors obtained by immunomagnetism, although final total and progenitor cell numbers are similar. Our results suggest that the populations separated by the two techniques differ mainly in the expansion capacity of progenitors and in the recovery of BFU-E after the selection procedure. These differences between two methods, which already are widely employed in research and in clinical transplantation, should be taken into account when considering the aims of the experiments.

Antigens, CD34↗
PubMed1979

Feulgen cytophotometry of pine nuclei. II. Effect of pectinase used in cell separation.

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.

Cell Separation↗
PubMed2005

[Collection of peripheral blood stem cells from ABO incompatible allogeneic donors by using blood cell separator].

To evaluate the yield of the blood cell separator for collection of peripheral blood stem cells (PBSC) from ABO major and (or) minor incompatible allogeneic donors and the feasibility of PBSC component infusion to the recipients without removal of erythrocytes or plasma, the Cobe Spectra (Version 6.1) blood cell separator was utilized to collect PBSC component from 9 allogeneic donors. Of all the donors, 4 were ABO major incompatible, 2 were minor incompatible and the other 3 were both major and minor incompatible to corresponding recipients. In each cycle, different amount of PBSC component was harvested, and the variable volume plasma chased the cells into the bag was adjusted according to the ABO incompatibility. The nucleated cell count, percentage of mononuclear cell, number of CD34(+) cell and percentage of viable cell (trypan blue excluding rate) in the component were detected. At the time of infusion, a series of protective measures to the renal function of recipients were taken. The results showed that apheresis was twice performed on these eight donors to collected enough PBSC for transplantation or cryopreservation, except one apheresis was enough for cell amount needed by transplantation, as the donor's body weight was much heavier than that of the recipient. Altogether 17 apheresises were performed, the mean yield of nucleated cells was 3.77 x 10(10), in which 97% to 99% were mononuclear cells (MNC). The harvested number of CD34(+) cell was 8.62 x 10(6)/kg. All the trypan blue exclusion rate was 100%. In ABO major incompatible or both major and minor incompatible component, there were 8 - 10 ml packed erythrocytes; in ABO minor incompatible component, there were 80 - 120 ml of plasma. These components were infused into the recipients without removal of erythrocytes or plasma and no haemolytic reaction was observed in any recipient, and their hematopoietic functions soon recovered. Results suggest that enough PBSC can be acquired by using blood cell separator Cobe Spectra (Version 6.1), with the modified separation factors, and the collected PBSC component can be safely infused into the ABO incompatible recipients without removal of erythrocytes or plasma.

ABO Blood-Group System↗
PubMed1998

Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting.

Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as clones. The separation of virgin mouse mammary luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting, their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1 cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types, two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression of differentiation-specific cytoskeletal markers in vitro.

3T3 Cells↗
PubMed1999

Comparative evaluation of procedures with a Baxter CS-3000 cell separator for collecting peripheral blood cells from children.

The safety and efficacy of apheresis using a newly developed procedure with a small volume separation container holder (SVSCH, Baxter) for the collection of PBSC from children with cancer were retrospectively compared with our historical experience with other procedures using different equipment and the CS-3000 plus cell separator. The procedures tested included the application of (a) specialized collection protocol 4 using the combination of an SVSCH and a small volume collection chamber (SVCC) (group A: 6 collections in 4 patients), (b) standard lymphocyte collection protocol 3 with GRANULO separation (G) and A-35 collection chambers (group B: 9 collections in 5 patients), and (c) modified collection procedure 1-120 with G and SVCC (group C: 7 collections in 3 patients). Although the retrospective nature of this study and the differences in the cohorts tested prevent a reliable analysis, the percent decrease in the peripheral platelet count after collection tended to be minimum in group A (11% +/- 5% versus 23% +/- 4% [B] or 17% +/- 4% [C]). Moreover, the largest number of CD34+ cells was collected in group A (8.7 +/- 9.4 x 10(5)/100 ml processed blood) compared with groups B (5.5 +/- 7.5 x 10(5)/100 ml processed blood) and C (4.0 +/- 2.8 x 10(5)/100 ml processed blood). These data suggest that a procedure incorporating SVSCH can be safely and effectively applied to small children and enables the selective collection of PBSC with less contamination by platelets, which is useful for subsequent cell processing.

Adolescent↗
PubMed1993

A constant flux system using the 'magnetic cell separator' (MACS) for highly efficient T-cell depletion.

A method for highly efficient T-cell depletion using a 'Magnetic Cell Separator' (MACS) is described. T-lymphocytes were depleted from peripheral blood samples of 37 patients with chronic lymphocytic leukemia (CLL). The flow speed in the MACS column was adjusted by a syringe-driver allowing a constant flow speed of about 100 microliters/min. The mean content of T-cells after depletion was 0.17% (S.D. +/- 0.27). The effectiveness of depletion was calculated to a mean 93.7%. There was no correlation between the proportion of CD3-positive cells before and after depletion.

CD3 Complex↗
PubMed1991

Increase in circulating colony-forming units-granulocyte-macrophage during large-volume leukapheresis: evaluation of a new cell separator.

Peripheral blood stem cell (PBSC) collection was evaluated in two groups of normal donors who underwent large-volume leukapheresis on a blood cell separator. In Group A (n = 10), a 3-hour leukapheresis was performed. An average of 11.8 L of blood was processed with a mean flow rate of 66 mL per minute and a collection rate of 3 mL per minute. The PBSC product contained a mean 1.4 x 10(10) mononuclear cells (MNCs) (lymphocytes and monocytes), 1.27 x 10(6) colony-forming units-granulocyte-macrophage (CFUs-GM), and an average hematocrit of 4 percent (0.04). Postapheresis blood counts showed significant reductions in MNCs (19%) and platelets (45%) (p less than 0.005). Twenty-four hours later, the MNCs had returned to preapheresis levels. The platelet count returned to baseline only after 7 days. Circulating CFUs-GM remained stable for 3 days after apheresis but were increased twofold by Day 7 after apheresis (p = 0.025). Varying the product hematocrit from 1 percent (0.01) to 13.3 percent (0.13) did not change the number of CFUs-GM collected per MNC. In Group B (n = 4), an average of 18.5 L of blood was processed with a mean flow rate of 94 mL per minute and a collection rate of 3 mL per minute. The PBSC product was collected as four sequential samples and assayed for MNCs and CFUs-GM. Total MNCs averaged 1.7 x 10(10) (an increase of 21% relative to Group A) and CFUs-GM averaged 3.08 x 10(6) (an increase of 143%). Mean MNCs did not vary significantly among the four samples. However, CFUs-GM collected per minute (relative to the first sample) did show 1.26-fold (p = 0.001), 1.86-fold (p = 0.011), and 2.52-fold (p = 0.04) increases in the second, third, and fourth samples. These data suggest that MNCs and committed progenitor cells are recruited during large-volume leukapheresis. Moreover, there is a twofold increase in circulating CFUs-GM 1 week after apheresis.

Adult↗
PubMed1992

Collection of peripheral blood mononuclear cells as a byproduct of plateletpheresis with two different blood cell separators.

Peripheral blood mononuclear cells (PBMC) were collected as a byproduct of plateletpheresis of normal blood cell donors using modifications to standard automated protocols on either the CS-3000 or Spectra blood cell separator machine. Comparison of the PBMC products obtained showed X +/- SD WBC yields of 5.3 +/- 3.4 vs. 3.8 +/- 2.0 x 10(9) with the CS-3000 and Spectra, respectively (P < .0001). The majority of the cells were lymphocytes, with 13-15% monocytes with both machines. Sixteen percent of the WBC collected with the Spectra, but only 1% of those collected with the CS-3000, were granulocytes. The CS-3000 PBMC product contained fewer RBC (0.2 +/- 0.1 x 10(11) vs. 2.4 +/- 0.6 x 10(11)) and more platelets (1.6 +/- 0.6 x 10(11) vs. 0.35 +/- 0.39 x 10(11)) in a smaller volume (40 +/- 14 ml vs. 229 +/- 37 ml) than the Spectra products. Comparison of the platelet collections harvested when PBMC were also collected to platelets harvested using standard procedures on the same machine showed no change in platelet, WBC, or RBC yields for the Spectra. A significant increase in mean WBC contamination from 40 +/- 56 x 10(7) to 112 +/- 205 x 10(7) and a small, but statistically insignificant, decrease in platelet yield from 4.1 +/- 1.2 x 10(11) to 3.9 +/- 1.8 x 10(11) was observed in the CS-3000 platelet collections when PBMC were harvested. There was no sustained change in donor lymphocyte counts and no change in acute donor side effects or time requirements when PBMC were collected.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Donors↗
PubMed1983

Identification of circulating maternal T and B lymphocytes in uncomplicated severe combined immunodeficiency by HLA typing of subpopulations of T cells separated by the fluorescence-activated cell sorter and of Epstein Barr virus-derived B cell lines.

Circulating maternal T cells were sought in a child with severe combined immunodeficiency (SCID) and no evidence of acute graft-vs-host disease, but who had small numbers (9 to 11%) of circulating T3-positive cells. HLA typing of unfractionated peripheral blood lymphocytes (PBL) and of isolated E rosette-forming cells (37 to 44% of PBL) failed to reveal the presence of maternal lymphocytes. T3-positive cells isolated by the fluorescence-activated cell sorter, however, expressed exclusively maternal HLA antigens. A lymphoblastoid B cell line established by infecting the patient's PBL with Epstein Barr virus then expressed exclusively maternal HLA antigens. The presence of maternal T and B cells in uncomplicated SCID may be more common than thought previously and calls for a careful assessment of the origin of any mature T cells that are present in affected infants. In addition, the presence of maternal cells in SCID may complicate the infant's therapy.

B-Lymphocytes↗
PubMed2005

Identification and molecular characterization of an N-acetylmuramyl-L-alanine amidase Sle1 involved in cell separation of Staphylococcus aureus.

We purified a peptidoglycan hydrolase involved in cell separation from a Staphylococcus aureus atl null mutant and identified its gene. Characterization of the gene product shows a 32 kDa N-acetylmuramyl-L-alanine amidase that we designated Sle1. Analysis of peptidoglycan digests showed Sle1 preferentially cleaved N-acetylmuramyl-L-Ala bonds in dimeric cross-bridges that interlink the two murein strands in the peptidoglycan. An insertion mutation of sle1 impaired cell separation and induced S. aureus to form clusters suggesting Sle1 is involved in cell separation of S. aureus. The Sle1 mutant revealed a significant decrease in pathogenesis using an acute infection mouse model. Atl is the major autolysin of S. aureus, which has been implicated in cell separation of S. aureus. Generation of an atl/sle1 double mutant revealed that the mutant cell separation was heavily impaired suggesting that S. aureus uses two peptidoglycan hydrolases, Atl and Sle1, for cell separation. Unlike Atl, Sle1 is not directly involved in autolysis of S. aureus.

Amino Acid Motifs↗
PubMed2002

Dielectrophoretic cell separation and gene expression profiling on microelectronic chip arrays.

Cell membrane dielectric properties of five different cultivated cell lines and human peripheral blood mononuclear cells (PBMC) were determined from dielectrophoretic crossover frequency measurements on a 5 x 5 microelectronic chip array. Based on distinct dielectric property differences between individual cell types, efficient cell separations were achieved by dielectrophoresis on this 5 x 5 array, which included separation of monocytic cells (U937) or human T cell leukemia virus type 1 (HTLV-1) tax-transformed cells (Ind-2) from PBMC, as well as separation of neuroblastoma cells (SH-SY5Y) from glioma cells (HTB). The purity of dielectrophoretically separated cells can be greater than 95%. Expression profiles of IL-1, TNF-alpha, and TGF-beta genes for U937 cells mixed with PBMC before and after the separation were determined by a means of electric field-facilitated hybridization on a 10 x 10 microelectronic chip array. By using the expression levels of pure U937 cells as a control, it was shown that the gene expression profiles of the postseparation cells were significantly different from those of the preseparation cell mixtures. The increase in gene expression levels for U937 cells upon lipopolysaccharide induction could be accurately determined only in the postseparation cells, while the preseparation samples masked these changes. Furthermore, by cultivating the separated HTB and SH-SY5Y cells and measuring expression of the stress-related gene c-fos, dielectrophoretic forces were shown to have little effect on cell survival and stress. The presented approach of using microelectronic chip arrays for both cell separation and gene expression profiling provides a great potential for accurate genetic analysis of specific cell subpopulations in heterogeneous samples.

Cell Line↗
PubMed2003

Comparison of two leukapheresis programs for computerized collection of blood progenitor cells on a new cell separator.

BACKGROUND: Peripheral blood progenitor cells (PBPCs) can be collected on various cell separators. Two leukapheresis programs (LP-MNC and LP-PBSC-Lym) were evaluated for computerized collection of PBPCs on a new cell separator. STUDY DESIGN AND METHODS: Leukapheresis assisted by the LP-MNC or LP-PBSC-Lym software was performed for the harvesting of PBPCs in 52 oncology patients after chemotherapy plus G-CSF treatment and in 18 healthy subjects after G-CSF mobilization alone. RESULTS: A total of 38 components from 33 donors via LP-MNC and 43 components from 37 donors via LP-PBSC-Lym were collected with a median of one (range, one to two) standard-volume leukapheresis procedures (9.2-13.3 L) per donor. There were no significant differences between the two groups concerning median counts of WBCs, CD34+ cells, CD34+ cell yields per harvest, and CD34+ cell yields of cumulative harvests. The blood cell counts after leukapheresis revealed that the LP-MNC resulted in significantly higher platelet loss than LP-PBSC-Lym (p = 0.024): 35.9 percent (range, 19.2%-66.1%) versus 29.7 percent (11.6%-52.3%). Regarding the CD34+ cell collection efficiency, the LP-MNC program was significantly better than the LP-PBSC-Lym program (p < 0.001): 77.5 percent (range, 35.5%-98.9%) versus 58.3 percent (range, 20.4%-98.9%). However, concentrates collected by the LP-PBSC-Lym program had significantly higher percentages of MNCs (p < 0.001) and CD34+ cells (p = 0.028) than harvests with the LP-MNC program: 90 percent (range, 69%-99%) versus 70 percent (range, 35%-98%) and 1.2 percent (range, 0.2%-7.3%) versus 0.7 percent (range, 0.2%-6.0%), respectively. No leukapheresis-related serious adverse events were seen, and time for hematopoietic engraftment was equivalent to data published in the literature. CONCLUSION: The LP-MNC program shows a significantly better CD34+ cell collection efficiency than the LP-PBSC-Lym program. However, collections with the LP-MNC program result in PBPC components with a lower MNC and CD34+ cell concentrations and a higher apheresis-related loss of patient's platelets.

Antigens, CD34↗
PubMed1979

Cell wall-bridge maintaining three dimensional structure of cell packets formed by the localized suppression of cell separation of a Micrococcus lysodeikticus (luteus) mutant.

Cell packets of Micrococcus lysodeikticus (luteus) mutant strain MT grown in medium supplemented with trypsin consisted of a tetrad as the unit structure. An interstice was observed between the unit-tetrads, and a three dimensional structure of cell packets was maintained by the cell wall-bridge along the rim of the cell packets which linked each unit-tetrad. This unique structure of strain MT cell packets seemed to occur when the cell separation was suppressed locally, i.e., when the cross wall inside the initial site of cell separation was cut off, while the wall outside the initial site of separation was not cut off but remained as a joint of the daughter cells. The mechanism of cell wall-bridge formation is discussed in connection with cell separation.

Amino Acids↗
PubMed1991

Plateletpheresis with the new Fresenius AS 104 blood cell separator.

Among the many blood cell separators introduced into the international market in these last few years, the Fresenius AS 104 represents an advanced and safe thrombocytapheresis machine whose development took advantage of extensive worldwide experience with blood cell separation. Nonetheless the AS 104 has generated most interest in West Germany and most, if not all, the studies published on its platelet collection efficiency have been carried out in that country. It is normally reported that from 2.7 to 3.5 x 10(11) platelets can be collected in approximately 80 minutes. Since these results could not be duplicated routinely in our hemapheresis unit, we set up a study by modifying the standard procedure. It was possible to reduce the procedure time and to collect platelet concentrates containing more than 4 x 10(11) cells on a routine basis by using the following procedure: ACD-A/blood ratio 1:10; Interface position 6:2; blood flow rate always exceeding 65 mL/min; rpm 1750; cell collection from 4 to 7 mL/min; volume of blood processed 3.6 L followed by the rinsing of the system with 200 ml of saline; extraction of the content of the secondary separation chamber by the action of the plasma pump working at 20 mL/min for 2 min. With this procedure the platelet yield in 34 collections exceeded 3.1 x 10(11) and averaged 4.06 x 10(11). The procedure time was reduced to 56.5 minutes with a mean blood flow rate of 62.3 mL/min. The leukocyte and erythrocyte contamination of the products were in the range of 1 x 10(7) and 1 x 10(8) respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation Studies as Topic↗
PubMed1998

Intrapatient comparison of an intermittent and a continuous flow cell separator for the collection of progenitor and stem cells from the blood.

BACKGROUND AND OBJECTIVES: Continuous-flow and intermittent-flow blood cell separators (CFCS and IFCS) are both used to collect stem cells from the blood to rescue patients undergoing myeloablative treatment for cancer. MATERIALS AND METHODS: We designed a study to compare the collection efficiency of the two systems. The continuous-flow Cobe Spectra and the intermittent-flow Haemonetics MCS-3P were used to collect cells on consecutive days from 9 patients mobilised with G-CSF with or without chemotherapy. Blood obtained before leukapheresis and the leukapheresis product were analysed for their content of red and white cells, platelets, CD34-positive cells, GM-CFC, CFC-E, and BFU-E. An extraction ratio was calculated. RESULTS: We found that the CFCS extracted about 4 times more mononuclear cells per unit time, 3 times more CD34-positive, and 4 times more clonogenic cells than the IFCS. The subject acceptability of the two systems was similar. CONCLUSION: The CFCS is a more efficient system for stem cell collection. IFCS requires a longer harvesting time for the same result.

Adult↗