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PubMed1982

Polyacrylamide microbeads with entrapped protein A from S. aureus for cell separation.

A technique was developed for the identification and separation of cells according to their surface antigenic markers, based on immobilization of protein A by entrapment in polyacrylamide microbeads. This method is further development of the previous techniques with SpA-containing S. aureus bacteria, but has the advantage of having less unspecific binding to the cells. We show that this technique might be a useful tool for the separation of cells, exemplified by separation of cells on Petri dishes coated with a monolayer of SpA-microbeads. The viability of separated cells was unaffected and adherent cells were easily removed by trypsin.

Acrylic Resins↗
PubMed2000

Methodological study of cell separation with domestic immunomagnetic beads.

To establish the method of cell separation with domestic immuomagnetic beads, three methods were investigated. Direct method, SPA method and Biotin-Avidin method were applied to separate cell strain Hut-78 and CD4 positive cells. Separation rate of strain Hut-78 was more than 90% in direct method. Detachment rate with papain was over 95%. Cell activity was well retained. SPA method and Biotin-Avidin methods were also effective, but the direct method was superior to the other two techniques. Before separated by the direct method, CD4 positive cells constituted 46.4% +/- 6.4% of mononuclear cells (MNC), but in eliminated suspension there was only 6.2% +/- 2.3% CD4 positive cells left. In the separated part, 80.6% +/- 7.2% of the cells combined with the beads. It is concluded that the direct method in separating cells had high sensitivity and specificity.

CD4-Positive T-Lymphocytes↗
PubMed1982

Rapid isolation of mononuclear cells from buffy coats prepared by a new blood cell separator.

A newly developed cell separator for the preparation and fractionation of buffy coat cells from human peripheral blood is described. In this cell separator buffy coats (BC-1) as routinely obtained from blood banks were used for the preparation of a second buffy coat (BC-2) with a volume of only 5-6 ml. A special fractionation device allowed sterile isolation of almost pure platelets and mononuclear cells with recoveries of 75 +/- 10% and 89 +/- 4% respectively. The white blood cell contamination of the platelet suspension never exceeded 20 X 10(6) leukocytes (i.e., less than 1 leukocyte per 5000 platelets). Furthermore, the mononuclear cell suspensions were shown to be contaminated with only 3 +/- 2% granulocytes, whereas the white blood cell/red blood cell ratio was 2.6 +/- 1.6, so that they could therefore be directly used for further separation by means of centrifugal elutriation. These results indicate that this cell separator provides a rapid (+/- 1 h) isolation of both platelets and mononuclear cells without exposing the buffy coat cells to foreign substances like Ficoll or Percoll.

Blood Platelets↗
PubMed1998

Effect of interface/offset (I/O) adjustment on collection efficiency using the Fenwal CS3000 Plus Blood Cell Separator for peripheral blood progenitor cell collection.

Peripheral blood progenitor cells (PBPC) reside within the mononuclear cell (MNC) component of the blood and can be collected using a number of apheresis devices, including the Fenwal CS3000 Plus Blood Cell Separator. Increased MNC collection efficiency, therefore, may reduce the number of apheresis required to achieve collection goals. In this study, patients were divided into groups by absolute MNC count to determine the effect of interface detector offset (I/O) adjustment on MNC collection efficiency. Apheresis products from 104 procedures collected using a standard I/O setting of 100 were compared with 121 collections for which the I/O setting was adjusted according to the preapheresis MNC count. Adjustment of the I/O setting in this manner had no statistically significant impact on the per kilogram dose of MNC collected. The data did show that MNC collection efficiency was reduced as both the MNC count and I/O setting increased, as the collection efficiency was greatest for patients with the lowest peripheral MNC counts and was inversely correlated with the preapheresis MNC count. Although contamination of the product with platelets was drastically reduced at higher I/O settings, there was a concomitant rise in RBC contamination. We conclude that a standard setting of 100 is preferable to adjustment of the I/O setting as a function of the preapheresis MNC count.

Adult↗
PubMed2006

Rapid separation and counting of viable microbial cells in food by nonculture method with bioplorer, a focusing-free microscopic apparatus with a novel cell separation unit.

A nonculture method utilizing a novel apparatus, the bioplorer, was developed. The bioplorer is composed of an efficient cell separation unit, a focusing-free microscopic device, and an image analysis program. A meat or vegetable suspension is poured into the cell separation funnel, and insoluble matter in the sample suspension is trapped by prefilters. Microbial cells passing through the two prefilters are then trapped by the membrane filter (pore size, 0.4 microm). Trapped cells are double-stained with 4',6'-diamidino-2-phenylindole and propidium iodide, and the membrane filter is removed and set on the focusing-free microscope. A fluorescent image is then recorded. Total numbers of viable and dead cells on the membrane filter can thus be determined automatically. One assay can be performed within 10 min, which is much faster than the culture method. The results obtained with both the nonculture method and the culture method for meat and vegetable samples were highly correlated (r = 0.953 to 0.998). This method is feasible for the practical purpose of food safety control.

Bacteria↗
PubMed2002

Effects of magnetic cell separation on monocyte adhesion to endothelial cells under flow.

Studies on monocyte adhesion are frequently limited by spontaneous changes of CD11b and CD62L during cell purification. Most isolation protocols for flow cytometric analysis that overcome this problem cannot be used when large numbers of living cells are needed for functional adhesion assays. This study investigated whether magnetic cell separation of monocytes with a paramagnetic bead against CD33 is a feasible method combining high yield with a low degree of spontaneous activation. As determined by flow cytometry, isolation of magnetically tagged monocytes at 4 degrees C did not alter the expression of CD11b and CD62L when compared to whole blood controls. Warming the cells slowly to room temperature immediately before starting the adhesion assay in a parallel plate flow chamber at 37 degrees C prevented further upregulation of adhesion molecules due to rewarming. When adhesion of magnetically tagged monocytes was compared with untouched monocytes that had been isolated via depletion of contaminating leukocytes, videomicroscopy showed that labelling CD33 neither affected rolling nor firm adhesion to human umbilical venous endothelial cells under flow. Finally, the subsequent upregulation of tissue factor expression on adherent monocytes indicates that magnetically separated monocytes responded properly to activating stimuli during cell adhesion. We conclude that magnetic cell separation via CD33 represents a feasible method for cell separation whenever large numbers of non-activated monocytes are needed for adhesion assays under flow.

Antigens, CD↗
PubMed2004

Recommendations for optimized settings of the Amicus Crescendo cell separator for the collection of CD34+ progenitor cells.

BACKGROUND: CD34+ PBPCs for autologous transplantation purposes are collected by leukapheresis procedures on automated cell separators. In this study, the influence of different parameters on collection efficiency (CE) of the Amicus Crescendo cell separator (Baxter) was investigated. STUDY DESIGN AND METHODS: A total of 146 PBPC collections with Amicus cell separators were performed in 56 patients with either settings recommended by the manufacturer or modified settings to identify variables that have a significant and important impact on CE. RESULTS: By use of a standard setting with a cycle volume of 1400 mL, CE significantly decreases when patients' preapheresis peripheral blood WBC counts are between 25,000 and 35,000 per micro L. CE can be improved if cycle volume is reduced to 1000 mL. If WBC concentrations exceed 55,000 per micro L before apheresis, CE also significantly decreases despite of reduced cycle volume. Additionally, high flow rates greater than 60 mL per minute significantly reduce CE. CONCLUSION: Parameters influencing the outcome of CD34+ PBPC collections were identified, such as patients' WBC count, cycle volume, and whole blood flow rate. An optimized adjustment of these variables will further increase the CE of the device.

Antigens, CD34↗
PubMed1993

Evaluation of platelet concentrates with the new Dideco blood cell separator: Excel.

The Excel Cell Separator produced by Dideco represents a good compromise between automation and Platelet efficiency. Platelet activation is a phenomenon that we can find with the concentrates of all cell separators. Although, the complement activation of Excel is extremely lower in comparison with the same data obtained using all the other cell separators, the 5-day storage is acceptable. It will be important to research the bacteriological controls. Like the pH of the platelet bags, because in certain cases it was too low. For the future it will be interesting to study the possibility to predict the quantity of platelets collected having a system that is adaptable with the donor parameters.

Blood Donors↗
PubMed2002

Controlled shear affinity filtration (CSAF): a new technology for integration of cell separation and protein isolation from mammalian cell cultures.

Controlled shear affinity filtration (CSAF) integrates animal cell separation and product isolation in a single unit operation through the use of a specifically designed rotating disk filter with incorporated membrane chromatography column. Because of the decoupling of shear force and pressure generation and the specific hydrodynamics of the system, shear rates can be easily optimized and precisely controlled to maximize filtration performance while viability of the shear sensitive animal cells is maintained. In this study, the general methodology is demonstrated using the integration of Chinese hamster ovary cell separation and isolation of recombinant tissue plasminogen activator (t-PA) as a model example. Direct capture of t-PA from cell culture broth was realized by using custom-made affinity membranes with lysine as a robust, small molecular weight affinity ligand. Small-scale t-PA adsorption experiments, as well as microfiltration experiments, were used to design the integrated CSAF process. A Chinese hamster ovary batch culture was processed with a lab-scale prototype, yielding 86% of the t-PA in the concentrated, particle-free eluate, whereas 95% of the bulk protein was removed. Because the viability of the cells is not significantly affected and high specific flux rates can be achieved, the CSAF technology should also be well suited for continuous perfusion with integrated product isolation. A truly continuous operation could be realized with two systems in tandem configuration.

Animals↗
PubMed1999

White cell reduction during plateletpheresis: a comparison of three blood cell separators.

BACKGROUND: White cell (WBC)-reduced single-donor platelet concentrates (SDPs) can be collected by the newest generation of blood cell separators. Three WBC-reduction techniques during plateletpheresis were investigated in the present study with respect to WBC content and platelet yield. STUDY DESIGN AND METHODS: The Amicus device used the elutriation principle for WBC reduction, and separations with periodically alternating interface position (PAIP) were employed in the AS.TEC 204. WBC reduction by in-line filtration was performed in the MCS+. Platelets were measured electronically and WBCs were determined manually (Nageotte chamber). RESULTS: In-line filtered SDPs showed significantly lower WBC content (0.088+/-0.178 x 10(6)) than SDPs that were WBC reduced by elutriation (0.31+/-0.48 x 106) or PAIP technique (0.89+/-1.57 x 10(6), p = 0.0001). Platelet yield (5.0+/-0.46 x 10(11)) was significantly higher in components obtained with the Amicus device (p = 0.0001). The AS.TEC 204 and MCS+ gave similar results for platelet yields: 3.15+/-0.63 and 3.28+/-0.71 x 10(11), respectively. CONCLUSIONS: The plateletpheresis systems studied allow the collection of WBC-reduced SDPs. In-line filtration resulted in the best WBC reduction. Some SDPs collected with the devices studied had a WBC content >1 x 10(6) per unit. Platelet yield was significantly higher in SDPs from the Amicus device.

Blood Cells↗
PubMed1980

Changes in the surface coat of mesenchymal cells of mouse limb buds after enzymatic cell separation.

Isolation of cells is nowadays performed by enzymatic means. The influence of such enzymes on the surface coat of mesenchymal and blastemal cells during the dissociation of limbs buds from 11-day-old mouse embryos was studied electron microscopically after staining with ruthenium red. EGTA or collagenase failed to bring about cell separation. The surface coat seemed to be unchanged after collagenase treatment. After EGTA an increase in extracellular filaments was observed. The proteases alpha-chymotrypsin, dispase II, papain, pronase P and trypsin (0.2%, 37 degrees C, 20 min) succeeded in completely dissociating limb buds. Apart from single granules, there was a detachment of the surface coat from the cells in all cases studied. Hyaluronidase led to only partial separation, but the detachment of the surface coat was almost complete, indicating a GAG-rich surface layer on these cells.

Animals↗
PubMed2004

Cell separation of hepatocytes and fibroblasts through surface-modified polyurethane membranes.

The separation of fibroblast cells (L929 cells) and hepatocytes was investigated by using unmodified and surface-modified polyurethane (PU) foaming membranes (pore size of 12 microm) by the incorporation of various functional groups. L929 cells permeated more readily than hepatocytes, and very few populations of hepatocytes (<5%) permeated through the membranes. This result was thought to be due to the smaller cell size of the L929 cells (5-10 microm) relative to the hepatocytes (15-30 microm). The larger hepatocytes were thought to plug the pores of the membranes. A good cell separation between L929 cells and hepatocytes was achieved when the cell mixture permeated through the negatively charged PU membranes. The negatively charged membranes were thought to enhance the permeation of L929 cells because of the electrostatic repulsion between the membranes and negatively charged cells. On the other hand, the hepatocytes were unable to permeate through the membranes because of the sieve effect caused by their large cell size. The separation of hepatocytes isolated from mice at different ages was also accomplished by permeating the cell mixture through unmodified and surface-modified PU membranes.

Animals↗
PubMed1990

Preservation of cytotoxic function during multi-cycle immunomagnetic cell separations of human NK cells using a new type of magnetic bead.

The isolation from human peripheral blood lymphocytes of natural killer (NK) cell populations by a novel magnetic cell sorting (MACS) procedure yielded large amounts of viable cells with active cytotoxic function. Non-adherent B cell-depleted lymphocytes were sequentially labelled with specific monoclonal antibodies, biotin-conjugated second antibody, FITC-conjugated streptavidin and biotin-conjugated magnetic particles (diameter 50-150 nm). In the magnetic field of a permanent magnet, positively labelled cells were retained on columns with a ferromagnetic matrix. When OKT3 was used for the depletion, 96-99% of the T cells were removed. The resulting non-labelled NK cell population contained 78-89% Leu11b+ and 87-96% Leu19+ cells. Magnetic retention of NK cells mediated by anti-Leu19 yielded about 81% and retention mediated by anti-Leu11b about 80% of total cells as determined by positive fluorescence. The resulting labelled and unlabelled cell subpopulations maintained their full NK activity as determined in 4 h cytotoxicity assays against human K562 tumor cells. The viability of non-labelled cells was fully preserved, whereas that of labelled cells slowly decreased with increasing numbers of preparative cycles. Furthermore, the ability of the isolated NK cells to show enhancement of their NK cytotoxicity after preincubation with IL-2 was maintained. The cytotoxic function of NK cells was also preserved when two or more MACS cycles using the same or different antibodies were carried out. The saving of time and the physiological condition of the isolated cells offer valuable advantages over FACS procedures.

Antibodies, Monoclonal↗
PubMed1999

[Methods of mobilizing hematopoietic cells, their collection using cell separation, purging--processing of pathologic cells and enrichment with CD 34+ cells--negative and positive selection].

Autological transplantation of bone marrow as well as hematopoietic and precursor cells obtained from peripheral blood with the use of cell apheresis is a therapy applied in the treatment of hematological diseases and solid tumours. The mobilization of hematopoietic cells is performed by applying cytostatic drugs and/or recombinant growth factors (G-CSF, GM-CSF). The collection of CD 34+ cells is performed by using cell separators. An important role in the transplantation procedure is played by purging techniques of transplantation material from residual neoplastic cells (negative selection) or isolation of hematopoietic cells (positive selection). The considerable progress in this field is connected with the implementation of Immunoadsorbtive or magnetic methods as well as those of molecular biology. The improvement of the procedure of hematopoietic cells transplantation and its efficacy is caused by the stimulation of the hematopoiesis by using the combination of cytokines (G-CSF, GM-CSF, IL-3, SCF) and purging of the hematopoietic cells obtained from the cellular apheresis.

Antigens, CD34↗
PubMed2000

Stem cell collection using the dideco excel continuous flow blood cell separator: parameters for optimal stem cell collection timing.

This study evaluates stem cell collection procedures performed with the Dideco Excel blood cell separator, with particular attention given to yields and separator collection efficiencies. Patients' blood precounts and yield parameters related to the harvest capacity of the collection system were investigated. Fifty-five collection procedures were analyzed in 32 patients suffering from hematological malignancies and solid tumors and mobilized with chemotherapy plus G-CSF. The median blood volume processed in each procedure was 15.8 liters (12-19.750), with a blood flow rate of 70 ml/min. Patients had the following median blood precount value: NC 7.81x10(9)/L, CD34+ cells 49.08x10(3)/ml. Leukapheresis procedures gave the following yields: NC 14.95x10(9), MNC 10.83x10(9), CD34+ cells 4.37x10(6); yields/kg, NC 0.21x10(9)kg, MNC 0. 15x10(9)/kg CD34+ cells 4.26x10(6)/kg. Procedures show the following collection efficiencies: NC 10.79%, MNC 29.06%, CD34+ 42.33%, PLT 26.5%. The RBC (red blood cell) contamination of the product was (median value) 20.9 ml for each procedure, and for platelets 1.76x10(11) per procedure. The CD34+ cell precounts strongly correlated with the CD34+ yields/kg (r=0.82. p=0.000). Furthermore the NC and MNC precounts correlated with the CD34+ yields/kg but only the MNC precount correlation is notable (r=0.57, p=0.000). The logistic regression analysis shows that CD34+ (p=0.008) but not NC (po=0.14), MNC (p=0.09), or PLT (p=0.53) precounts significantly influenced the collection of a sufficient dose of CD34+ cells for transplantation (> or =2.5x10(6)/kg). Eleven of the thirty-two patients have been transplanted till now, and all had a prompt and lasting trilineage engraftment NC >1x10(9)/L on day 12 (10-17). Our data show that the collection system analyzed in this report is able to collect large amounts of progenitor cells, harvesting >2.5x10(6)/kg CD34+ cells with a single procedure in 68.8% of patients and assuring complete recovery after stem cell transplantation.

Adolescent↗
PubMed1995

Comparison of Cobe Spectra and Haemonetics MCS-3P cell separators for peripheral blood stem cell harvesting.

A prospective study was undertaken to compare the total nucleated cell (TNC), mononuclear cell (MNC), CD34+ cell, and CFU-GM yields of two different cell separators. A Haemonetics MCS-3P and a Cobe Spectra machine were used to leukapherese 10 patients with malignant diseases on 4 consecutive days after mobilization with G-CSF. All patients were harvested twice on each machine for a fixed period of time. The blood volume processed (10.47 vs 3.79 l, P < 0.001), MNC yield (2.66 vs 0.90 x 10(8)/kg; P < 0.001), MNC yield rate (1.66 vs 0.55 x 10(6)/kg/min; P < 0.001), MNC purity (81 vs 42%; P < 0.001), CFU-GM yield (18.1 vs 5.5 x 10(4)/kg; P = 0.001), and CFU-GM yield rate (11.27 vs 3.42 x 10(2)/kg/min; P = 0.001) were significantly higher with the Spectra. The CD34+ cell yields and yield rates were comparable. Although CFU-GM and MNC yields per unit blood volume processed were comparable for both machines, there was a trend for higher CD34+ yields per unit volume processed with MCS-3P. We conclude that Spectra is faster than MCS-3P with more blood processed per unit time resulting in higher cell yields, but yields per unit volume processed are comparable for both machines. The choice of machine for a given patient depends upon convenience, venous access and the time available.

Adult↗
PubMed2006

Factors affecting volume reduction and red blood cell depletion of bone marrow on the COBE Spectra cell separator before haematopoietic stem cell transplantation.

The COBE Spectra is used to volume/red blood cell (RBC) deplete BM before transplantation or cryopreservation. We have audited our results to identify the effect of transit time, refrigerated storage, age and cellular composition on mononuclear cells (MNC) and CD34+ cell recoveries, volume/RBC depletion and neutrophil engraftment. In total, 88 consecutive collections from autologous (n = 25) and allogeneic (n = 63) donors were included. The mean collection volume was 1250 +/- 398 ml with RBC content of 341 +/- 113 ml. The MNC and CD34+ cell recoveries were 83.3 +/- 18.5 and 88.1 +/- 18.9%, respectively, volume depletion was 88.2+/-4.4% and RBC depletion 98.3 +/- 1.8%. Neutrophil engraftment was achieved in 20.1 +/- 6.4 days. Factors affecting MNC and CD34+ cell recoveries were transit time (P = 0.0060), overall age (P < 0.0210) and MNC/CD34+ cell concentrations (P < 0.0313). The presence of crenated RBC also reduced CD34+ cell recovery (P = 0.0028). Refrigerated storage did not adversely affect cell recovery (P > 0.8161) or neutrophil engraftment (P = 0.8959). This study demonstrates that time in transit, overall age, MNC and CD34+ cell concentrations and RBC condition were important factors affecting processing. RBCs show artefacts soon after collection at ambient temperatures and these may interfere with the separation and collection of MNC/CD34+ cells. Refrigeration at 4-6 degrees C during transit and storage may reduce formation of RBC artefacts and maximize MNC and CD34+ cell recoveries.

Antigens, CD34↗