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At least 199 records · Page 11Linked to original sources
PubMed1987

Polyploidization of G2M phase cells separated from aerobically and anaerobically grown Ehrlich ascites tumor cells.

G2-enriched fractions of Ehrlich ascites tumor cells (up to 80%-85% G2 cells) separated from anaerobically and aerobically cultured asynchronous populations by centrifugal elutriation revealed the same growth characteristics after recultivation under standard conditions: a significant proportion of cells with increased DNA (DNA content greater than 4C) emerged. Interruption of DNA synthesis by deprivation of oxygen may account for polyploidization (over-replication) of DNA but other mechanisms must be taken into consideration.

Aerobiosis↗
PubMed1993

Evaluation of a novel automated protocol for the collection of peripheral blood stem cells mobilized with chemotherapy or chemotherapy plus G-CSF using the Fresenius AS104 cell separator.

Forty-seven peripheral blood stem cell (PBSC) collections were carried out on patients mobilized with chemotherapy and 63 on patients mobilized with chemotherapy plus G-CSF (Filgrastim), using the Fresenius AS104 cell separator and a novel automated PBSC collection protocol. As expected, cell yields were significantly higher in the series mobilized using chemotherapy plus G-CSF. The low platelet and red blood cell contamination permitted freezing of the apheresis product without further manipulation, other than plasma removal in both series. In patients mobilized with chemotherapy we obtained a MNC and a hemopoietic progenitor (CFU-GM, BFU-e, and CD34+ cells) collection efficiency comparable or superior to those reported by Bender (1992) with the Baxter CS3000 Plus after mobilization with cyclophosphamide. A significant decrease in MNC, BFU-e, and CD34+ cell collection efficiency was found in patients mobilized with chemotherapy plus G-CSF compared to those obtained in patients mobilized with chemotherapy alone. Ten patients achieved a prompt and stable engraftment after high dose chemotherapy and the infusion of cryopreserved PBSC collected using this protocol. Studies are in progress in order to improve MNC and hemopoietic progenitor collection efficiency in patients mobilized with G-CSF to obtain a graft in no more than one or two procedures.

Adolescent↗
PubMedDate not supplied

Collection of platelets depleted of red and white cells with the "surge pump" adaptation of a blood cell separator.

The surge pump is a device which modifies platelet collection of a blood cell separator so that red and white blood cell contamination is minimized. Plasma collected from the donor is directed back into the centrifuge bowl at 200 ml per min, where it causes platelets to be floated off the red cell-plasma interface and thus is collected as an almost pure platelet preparation. Fifty plateletapheresis procedures with the surge pump adaptation were compared to 50 procedures using the standard red cell method. Mean (+/- SD) white cell (greater than 95% lymphocytes) contamination was 5.4 +/- 3.1 X 10(8) cells per collection with the surge pump and 63.5 +/- 10 X 10(8) cells per collection with the standard red cell method (p less than 0.0001). Mean collection hematocrit was 8.1 +/- 2.6% with the standard method and less than 1% with the surge pump eliminating the need for crossmatch or centrifugation to remove red cells from ABO incompatible platelets. Surge pump collection produced a mean of 4.0 +/- 1.6 X 10(11) platelets compared to 5.0 +/- 2.0 X 10(11) platelets for the standard method (p less than 0.01). The mean time per run was 14.8 +/- 2.4 min with the surge pump compared with 18.1 +/- 3.3 min with the standard method (p less than 0.001). Therefore, the platelet yield per minute of procedure time was comparable with both methods (surge pump, 37.3 +/- 11.7 X 10(8) platelets per min; standard method, 39.2 +/- 14.3 X 10(8) platelets per min.). Surge pump operation was learned easily by technologists and caused no donor complications. The surge pump is a simple and effective way of minimizing white and red blood cell contamination in platelet collections from the blood cell separator studied without compromising platelets yields.

Blood Cell Count↗
PubMed1975

Cell separation by staphylococcal protein A-coated erythrocytes.

A method for cell separation according to cell surface markers is described. Heat-aggregated human IgG or IgG antibodies, raised to surface markers of mouse lymphocytes-for example, Ig or theta antigen-are allowed to react with the lymphocytes. When these cells are incubated with sheep erythrocytes coated with protein A of Staphylococcus aureus, rosettes are formed. These rosettes can be separated from nonrosetted lymphocytes by density gradient centrifugation. The erythrocytes of the rosettes are disrupted by complement action or by osmotic shock, and the lymphocytes are recovered. Immunoglobulin-protein A complexes are shed off the cell surfaces by cultivation. In this way cells with, as well as those without, surface markers could be obtained with high purity (up to 80%) and with high viability.

Animals↗
PubMed1999

Purging of contaminating breast cancer cells from hematopoietic stem cell grafts by adenoviral GAL-TEK gene therapy and magnetic antibody cell separation.

The presence of contaminating tumor cells in autologous bone marrow or peripheral blood stem cell (PB-SC) preparations increase the likelihood of relapse in women receiving transplants for metastatic breast cancer. We describe a new technique for purging breast cancer cells (BCCs) that combines two independent strategies: (a) the specific enrichment of CD34+ progenitor stem cells by magnetic antibody cell separation (MACS), and then (b) infection of the contaminating BCCs with a recombinant adGAL-TEK marker/suicide gene adenovirus (ad-v), followed by the addition of ganciclovir (GCV). Infection with this ad-v results in three to four times greater expression of ad-v-delivered reporter gene in BCCs than in CD34+ cells. In addition -2 h, -low multiplicity of infection (50:1) adGAL-TEK infections of BCC lines (MCF-7 and BT474) eradicated >99% of BCCs after 72 h of exposure to 20 microM GCV. However, exposure to both adenovirus and GCV at the MOIs and doses used had little effect on hematopoietic stem cells to form colonies in colony-forming unit assays. adGAL-TEK infection in our model system (10(3)-10(5) BCCs added into 10(7) HSCs) also resulted in the 3 to 5 log eradication of clonogenic BCCs after the addition of GCV. MACS enrichment/purification of CD34+ cells from PB-SC contaminated with 2 x 10(6) to 5 x 10(7) BCCs followed by adGAL-TEK infection and GCV addition resulted in 5-7-log depletion of clonogenic BCCs as well as enrichment of CD34+ progenitor cells to >98%, with the recovery of >70% of hematopoietic stem cells. This adenoviral purging system is so robust that poor MACS purification, resulting in 1.5-log depletion of BCCs, still permits excellent ad-v infection and BCC killing.

Adenoviridae↗
PubMed1979

Studies on a fractionated murine fibrosarcoma: proliferative potential of the separated cells.

A transplantable methylcholanthrene-induced fibrosarcoma of female BALB/c mice (the MC-2 fibrosarcoma) was dissociated by combined mechanical and enzymatic means, then fractionated by isopycnic centrifugation in linear albumin gradients. In some experiments recovered cells were both cultured in soft nutrietn agar and inoculated subcutaneously into syngeneic recipients. In these experiments a highly significant correlation was observed between subsequtnt colony number and rapid growth phase tumor size suggesting identity of clonigenic and tumorigenic cells. It was consistently found that clonigenic cells were markedly depleted from the low density extremes of the cell density distribution profiles suggesting that the low density neoplastic cells had irreversibly left the growth fraction. With increasing tumor age, sequential studies showed that both total and clonigenic cell density distribution profiles were variable, showing no obvious trend, suggesting that in the age (13-35 days) and size (2-8 g) range studied growth fraction changes had little selective effect on cells of any specific density. These results imply that a marked selective depletion of low density clonigenic cells (or selective accumulation of low density non-proliferative cells) must mainly occur during an earlier phase of tumor growth. Studies on several other murine solid tumors also showed maximal depletion of clonigenic cells from the least dense fractions, suggesting that this situation may be common.

Animals↗
PubMedDate not supplied

Optimization of yield in magnetic cell separations using nickel nanowires of different lengths.

Ferromagnetic nanowires are shown to perform both high yield and high purity single-step cell separations on cultures of NIH-3T3 mouse fibroblast cells. The nanowires are made by electrochemical deposition in nanoporous templates, permitting detailed control of their chemical and physical properties. When added to fibroblast cell cultures, the nanowires are internalized by the cells via the integrin-mediated adhesion pathway. The effectiveness of magnetic cell separations using Ni nanowires 350 nm in diameter and 5-35 micrometers long in field gradients of 40 T/m was compared to commercially available superparamagnetic beads. The percent yield of the separated populations is found to be optimized when the length of the nanowire is matched to the diameter of the cells in the culture. Magnetic cell separations performed under these conditions achieve 80% purity and 85% yield, a 4-fold increase over the beads. This effect is shown to be robust when the diameter of the cell is changed within the same cell line using mitomycin-C.

3T3 Cells↗
PubMed1991

Cibacron blue 3G-A inhibits cell separation of gram-positive bacteria.

A triazine dye, Cibacron blue 3G-A (CB), is an inhibitor of cell separation of staphylococcal spp. therefore, we examined the effect of CB on growth of gram-positive bacteria other than Staphylococcus. CB added to the medium of growing cultures of strains of genus Micrococcus, Streptococcus, Lactobacillus and Bacillus caused inhibition of cell separation. Moreover, in case of Bacillus and Lactobacillus, individual cells were elongated as filament. Strains of the genus Micrococcus were as sensitive to CB as genus Staphylococcus in which the minimum concentrations of CB needed for inhibition of cell separation ranged from 15 to 100 microM. Other strains belong to genus Streptococcus, Bacillus and Lactobacillus were less sensitive; the minimum concentrations were 100 microM--25 mM.

Bacillus↗
PubMed1998

Differential induction of P-selectin expression on platelets by two cell separators during plateletpheresis and the effect of gender on the release of soluble P-selectin.

BACKGROUND: Though a number of studies have elegantly characterized platelet activation during storage, less attention has been paid to the initial activation caused by different collection procedures. STUDY DESIGN AND METHODS: The effects of two blood cell separators on the initial activation of platelets were measured by flow cytometric analysis of P-selectin expression in 13 male donors on one cell separator (CS 3000 Plus) and 11 men and 9 women on the other (MCS 3P). In addition, the storage and release of soluble P-selectin (circulating P-selectin [cP-selectin]) by platelets were quantified, to determine whether the change in cP-selectin is a more sensitive marker for initial platelet activation, and the influence of gender on measured endpoints was evaluated. RESULTS: The CS 3000 Plus increased the percentage of P-selectin-positive platelets from a median of 3.4 percent before apheresis to 7.6 percent (p = 0.006) in platelet concentrates (PCs), whereas the MCS 3P did not (p = 0.002 between the two cell separators). When preapheresis cP-selectin levels were compared to those in apheresis PCs, cP-selectin increased from 51 to 101 ng per mL in plasma of CS 3000 Plus PCs, whereas cP-selectin levels increased from 53 to 78 ng per mL in MCS 3P PCs (men) and from 48 to 99 ng per mL in MSC 3P PCs (women) (p<0.005 for all). The relative increase in cP-selectin was higher in women than in men in MCS 3P PCs (p = 0.013). Concomitantly, the amount of P-selectin stored in platelets before apheresis decreased (p<0.025 for all). When donors undergoing apheresis on the MCS 3P were compared, the amount of P-selectin stored in the platelets of PCs was higher in men than women (p = 0.026). CONCLUSION: This trial shows 1) that initial activation of platelets obtained with the MCS 3P is less than that of platelets obtained with the CS 3000 Plus; 2) that the increase in cP-selectin is a more sensitive marker for initial platelet activation than the expression of P-selectin on the surface; and 3) that the relative amount of cP-selectin is higher in women than in men given the same stimulus. Differences in platelet activation by various cell separators and the sex of the donor may contribute to variability of PC quality.

Blood Platelets↗
PubMed1994

Peripheral blood stem cell collection using the small volume collection chamber in the Fenwal CS-3000 Plus blood cell separator.

Optimal methods for peripheral blood stem cell (PBSC) collection should yield a small volume product containing minimal platelets and a large number of mononuclear cells (MNC). The Fenwal CS-3000 Plus blood cell separator was modified in an attempt to meet these objectives. Modifications of the CS-3000 Plus included use of the small volume collection chamber (SVCC), increasing the interface/offset detector setting to 150 and decreasing the centrifuge speed to 1400 rpm. Thirty-eight patients undergoing 224 PBSC collections were studied. Mobilization methods included 4 g/m2 cyclophosphamide (CY), CY + 250 micrograms/m2 subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF) or GM-CSF alone. The median collection volume was 58 ml containing a median of 21 ml of red blood cells. Platelet collection efficiency was < 4.4% and the median number of extracted platelets was 0.6 x 10(11)/apheresis. Median reduction in the platelet count post-apheresis was 15%. MNC purity was 95.5% and MNC collection efficiency was 61%. Yield of MNC was 1 x 10(8)/kg/apheresis. Collected progenitor cells correlated with both the WBC and MNC content of the apheresis product. The modified CS-3000 Plus with the SVCC is effective for PBSC collection following three different mobilization regimens and is, therefore, recommended for routine collection of PBSC.

Adult↗
PubMed1994

[Results of cell separation with new single needle techniques in comparison with a current dual needle technique].

BACKGROUND: In plateletpheresis, new single-needle techniques have been introduced for the cell separators A 201 and AS 104. These techniques were compared with the well established CS 3000 Plus cell separator, which uses the continuous flow system. MATERIAL AND METHODS: Donation time, ACD consumption, platelet yield, separation efficiency, erythrocyte and leukocyte contamination were studied in different protocols with the dual- and single-needle procedure. RESULTS: Total amount of thrombocytes and separation efficacy were lowest with the A 201 device. The separation efficiency was best with the single-needle version of the AS 104 separator due to reprocession of the blood during the return cycle to the centrifuge. Leukocyte contamination was lowest with the standard procedure of the CS 3000 Plus device. CONCLUSIONS: The single-needle technique of the AS 104 blood cell separator resulted in sufficient platelet collection. The purity of the platelet concentrates collected with the A 201 and AS 104 machine has to be improved.

Cell Separation↗
PubMed2004

Enrichment of rare cancer cells through depletion of normal cells using density and flow-through, immunomagnetic cell separation.

OBJECTIVE: To develop a reliable technique to enrich for rare cells in blood suspensions using only negative selection steps including a flow-through immunomagnetic cell separations system and by optimizing variables normally encountered during such enrichment processes. METHODS: A human breast cancer cell line was cultivated and spiked at a ratio of 1 cancer cell to 10(5) total leukocytes in buffy coat or 1 cancer cell to 10(8) total cells in whole blood samples. The final, optimized process consisted of: a red cell lysis step, immunomagnetically staining leukocytes with an anti-CD45 PE, anti- MACS sandwich, immunomagnetic sorting using a flow-through system (QMS), and a final cell analysis step using either an automated cell counter, filtration, and visual counting or a cytospin analysis. RESULTS: The final, optimized process produced a final enrichment of the rare cancer cells of 5.17 log(10) and an average, final recovery of 46%. It should be noted that a negative depletion protocol was used (i.e., no labeling of the rare cancer cells was used). CONCLUSIONS: To the authors' knowledge, no examples in the literature exist of a 5.17 log(10) enrichment of cancer cells in human blood using a negative depletion protocol. The closest example is a 4 log(10) enrichment in which two positive magnetic cell separation steps were used (none were used in this study). Ongoing studies are investigating further modifications of the precommercial, prototype flow-through immunmagnetic separation system to increase both the enrichment and recovery rate. However, even at current performance levels, the presented process could significantly improve visual and molecular analysis of rare cells in blood.

Antibodies, Monoclonal↗
PubMed1990

Plateletapheresis with the new cell separator AS-104.

Summarizing several studies, it can be shown that the new cell separator AS-104 (Fresenius), in comparison to the older systems, provides a real advantage concerning the separation efficiency, the purity of the platelet concentrates (PC), the platelet function and the donor safety. The results of 5 different separation protocols demonstrate the technical flexibility and developmental potential of this system. From the experience of 580 plateletapheresis runs the successful improvement of the cell separator, and the disposables can be documented as the frequency of disturbances and discontinuations are reduced considerably. The preliminary results of an uncontrolled, not randomized transfusion study of 244 transfusions in 49 patients show comparable posttransfusion increments as obtained with PC of the CS-3000 (Baxter).

Blood Transfusion↗
PubMed1983

Fetal calf serum augmentation during cell separation procedures accounts for the majority of human autologous mixed leukocyte reactivity.

In the present experiments, we have observed that cells separated using techniques that provide no xenoantigen exposure to stimulator cells do not participate in the autologous MLR. However, exposure of separated cells in general and stimulator cells in particular to fetal calf serum does lead to autologous MLR. These data confirm the previous observations of Huber et al. that the human autologous MLR is primarily due to xenoproteins absorbed to stimulating cells during the separation procedures and indicate that a reassessment of human autologous MLR studies will be needed.

Animals↗
PubMedDate not supplied

Effect of hydroxyethyl starch on platelet function following granulocyte collection using the continuous flow cell separator.

Hydroxyethyl starch (HES) was used as sedimenting agent in the course of granulocyte collection on seventeen normal donors, using the continuous flow cell separator (CFCS). No clinical bleeding was noted in any of the donors. In spite of a 21 per cent mean reduction in postcollection platelet count, all donors had platelet counts within normal limits precollection and postcollection. Postcollection platelet adhesiveness increased in five donors, remained the same in one donor and declined in ten donors. Except in one donor there was no change in previously normal platelet aggregation to adenosine diphosphate or collagen. In the dose used, 500 ml, the infusion of 6% hydroxyethyl starch as an adjunct to granulocyte separation using the continuous flow cell separator does not appear to adversely affect platelet number and function.

Blood Cell Count↗
PubMed1979

Density gradient centrifugation of cells separated from multicellular tumor spheroids.

Cells from a murine fibrosarcome (FSa) have been grown in vitro as multicell tumor spheroids (MTS). The growth rate of these MTS was determined. Following selected periods of growth, MTS were made into a single cell suspension and separated on linear density gradients of Renografin. While only 1 population of cells were separated from small spheriods (400 mum diameter), at least 3 subpopulations of tumor cells were separated and isolated from large spheroids (800 mum in diameter).

Animals↗
PubMedDate not supplied

Collection of peripheral blood stem cells using an automated discontinuous flow blood cell separator.

Twenty collections of peripheral blood stem cells were performed in 3 patients (2 NHL, 1 AML) using the Haemonetics V50S discontinuous flow blood cell separator. A modified lymphocyte collection protocol (Nebraska Surge) was used in all instances. Leukapheresis were performed after 1 or 2 courses of chemotherapy and started when peripheral blood leukocytes count reached 1 x 10e9/l and platelets count 80 x 10e9/l. A mean blood volume of 5.9 +/- 0.6 litres was processed per procedure and the mean yields for mononuclear cells, nucleated cells and CFU-GM were respectively 5.4 +/- 1.4 x 10e9, 4.9 +/- 1.6 x 10e9 and 128.5 +/- 182.3 x 10e4 per procedure. Haemonetics V50S had showed a mean collection efficiency for mononuclear cells of 67.5 +/- 5.0% per procedure. Results obtained are not significantly different from the ones obtained with an automated continuous flow separator even if extracorporeal circulation is consistently high in patients with a low hematocrit when the 250 ml Latham Bowl is used.

Cell Separation↗