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PubMed2005

Paramagnetic capture mode magnetophoretic microseparator for high efficiency blood cell separations.

This paper presents the characterization of continuous single-stage and three-stage cascade paramagnetic capture (PMC) mode magnetophoretic microseparators for high efficiency separation of red and white blood cells from diluted whole blood based on their native magnetic properties. The separation mechanism for both PMC microseparators is based on a high gradient magnetic separation (HGMS) method. This approach enables separation of blood cells without the use of additives such as magnetic beads. Experimental results for the single-stage PMC microseparator show that 91.1% of red blood cells were continuously separated from the sample at a volumetric flow rate of 5 microl h-1. In addition, the three-stage cascade PMC microseparator continuously separated 93.5% of red blood cells and 97.4% of white blood cells from whole blood at a volumetric flow rate of 5 microl h-1.

Algorithms↗
PubMed2001

Ex vivo evaluation of PBMNCs collected with a new cell separator.

BACKGROUND: This study reports on an evaluation of the ability of a cell separator (Amicus, Baxter Healthcare) and the integral MNC computer software program to collect a variety of MNC subsets. The collection efficiency (CE) of the Amicus for these MNC subsets was compared to that of another cell separator (CS-3000 Plus, Baxter). The collected MNCs were also assayed ex vivo to determine if these cells remained functional. STUDY DESIGN AND METHODS: Healthy volunteer blood donors were recruited to provide PBMNCs for the isolation of CD3+, CD4+, CD8+, CD19+, NK, and gammadelta+ cells and monocytes. Cells were collected with an Amicus (test arm; n = 16) or a CS-3000 Plus (control arm; n = 11) cell separator. Cells were counted on a flow cytometer and CEs were calculated. For functional studies, the Amicus-collected MNC data were compared to CS-3000 Plus historical data. Functional studies performed included surface antigen expression assays (CD8+), proliferation assays (CD4+ and CD8+ cells), NK cytotoxicity assays for K562 and HUVE cells, and E-selectin induction on endothelial cells through NK+ contact dependency. Dendritic cells (DCs) were generated from CD34+ cells collected on the Amicus, positively selected by the use of antibody-bound, magnetic bead technology, and then cultured ex vivo with a combination of growth factors to generate the DCs. RESULTS: CEs were higher on the Amicus than on the CS-3000 Plus for CD3+ (68 vs. 54%), CD4+ (70 vs. 56%), CD8+ (68 vs. 52%), and CD19+ (60 vs. 48%) cells (p<0.05). For the two separators, CEs were equivalent for monocytes, NK+, and gammadelta+ cells. The Amicus separator collected significantly fewer platelets than did the CS-3000 Plus (p<0.00001). CD4+, CD8+, and NK cells proliferated normally. NK cells appropriately stimulated E-selectin expression on endothelial cells. Culture-generated DCs obtained by using Amicus-collected CD34+ cells expressed appropriate cell surface markers. CONCLUSION: The Amicus separator is acceptable for the collection of PBMNC subsets. The device collects CD3+, CD4+, CD8+, and CD19+ T- and B-cell subsets with greater efficiency and collects MNCs with significantly fewer contaminating platelets than does the CS-3000 Plus. Cells collected on the Amicus are suitable for use in a variety of research and clinical immunobiologic studies.

Blood Donors↗
PubMedDate not supplied

The impact of parental death versus separation from parents on the mental health of Israeli adolescents.

Early research suggested that the loss of a parent during childhood can lead to depression and other psychopathology in children and adults. More recent research has differentiated between loss due to death and separation from parents and has questioned the link between early parental death and psychopathology. We examined the hypothesis that separation from parents has a more detrimental effect than the death of a parent on the mental health of adolescents in the community. Israeli adolescents (N = 844) with a mean age of 16.7 +/- 1.0 years (mean +/- SD) participated in this study. Seventy reported that a parent did not live at home during one of three 5-year periods until they reached the age of 15; 37 reported the death of a parent and 777 were from intact families. All participants completed the Brief Symptom Inventory (BSI), General Well Being Schedule (GWB), Parental Bonding Instrument (PBI), and Perceived Social Support Family/ Friends Scales (PSS-Fam and PSS-Fr). Adolescents who had experienced separation from parents had more psychiatric symptoms (BSI), expressed a lower sense of well-being (GWB), experienced less support from their family (PSS-Fam), and felt less cared for and more controlled by their parents (PBI) compared with those belonging to intact families. The death of a parent was not associated with significantly different scores on these variables. When we controlled for parental bonding (PBI) and perceived social support from family (PSS-Fam), the differences between the separation group and the intact family group were no longer significant. The psychological impact of separation from parents involves a greater risk for psychopathology than the death of a parent. The quality of the relationship with the parents moderates the negative impact of separation from them.

Adolescent↗
PubMed1979

Sebaceous gland differentiation: II. The isolation, separation and characterization of cells from the mouse preputial gland.

Viable cells have been isolated, by enzyme digestion, from mouse preputial glands and have been separated, by isopycnic centrifugation, into populations of different buoyant densities. The separated cells have been evaluated by morphological and biochemical criteria to assess the success of the separation procedure in terms of the stage of differentiation of each cell population. Use of Ficoll as gradient medium for the isopycnic centrifugation proved unsuccessful, but good separations were obtained when Metrizamide was used. The separated cells from the Metrizamide gradient were shown to be in different stages of differentiation. Techniques are described for the special handling of these cells as well as suitable assay procedures. Some of the properties of the separated cells are described.

Animals↗
PubMed2006

Separation of yeast cells from aqueous solutions using magnetically stabilized fluidized beds.

AIMS: To separate Saccharomyces cerevisiae cells from aqueous solutions using magnetically stabilized fluidized beds (MSFB) that utilize a horizontal magnetic field, and to study the effect of some parameters, such as bed porosity and height, liquid flow rate and inlet concentration on cell removal efficiency and breakthrough curves. METHODS AND RESULTS: The separation process was conducted in an MSFB under the effect of horizontal magnetic field. The magnetic particles used consist of a ferromagnetic core of magnetite (Fe3O4) covered by a stable layer of activated carbon to adsorb the yeast cells from the suspension. The yeast cell concentration in the effluent was determined periodically by measuring the absorbance at 610 nm. The effect of the magnetic field intensity on the bed porosity and consequently the exit-normalized cell concentration from the bed was studied. It was found that bed porosity increased by 75%, and the normalized cell concentration in the bed effluent decreased by 30%, when the magnetic field intensity was increased from 0 to 110 mT. In addition, increasing the magnetic field intensity and bed height delayed the breakthrough point, and allowed efficient cell removal. These results demonstrate an improved method to separate cells of low concentration from cell suspension. CONCLUSIONS: This study allows the continuous separation of yeast cells from aqueous solutions in an MSFB. The removal efficiency is affected by different parameters including the bed height, flow rate and initial concentration. The removal efficiency reaches 82%, and could be improved by varying the operational parameters. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this investigation show that the MSFB using horizontal fields represents a potential tool for the continuous separation of cell suspension from aqueous solution. This study will contribute to a better understanding of the hydrodynamic parameters on the separation efficiencies of the cell.

Cell Separation↗
PubMed2006

Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis.

Short tandem repeat (STR) analysis is a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. The feasibility of applying laser microdissection (LMD) technology to precisely separate sexual assault cell mixtures by visual inspection coupled with laser dissection was assessed through three experiments. First, various histological stains were evaluated for use with LMD and DNA analysis. Second, different DNA isolation methods were evaluated on LMD-collected cells. Finally, STR analysis was performed on LMD-separated sperm cells from mixtures of semen and female buccal epithelial cells. The results indicated (a) hematoxylin/eosin staining performed best in its ability to differentiate sperm and epithelial cells while exhibiting the least negative effect on further downstream analysis; (b) both QIAamp and Lyse-N-Go methods were useful for recovery of DNA from LMD-collected sperm cells; and (c) LMD separation provided clear STR profiles of the male donor with the absence of any additional alleles from the female donor. This report describes an efficient, low-manipulation LMD method for the efficient separation of spermatozoa from two-donor sperm/epithelial cell mixtures.

Alleles↗
PubMed1993

Adults who feared school: is early separation anxiety specific to the pathogenesis of panic disorder?

Although juvenile separation anxiety disorder is maintained to be a predisposing factor to adult panic disorder in DSM-III-R, past research has failed to clarify (a) whether it is separation anxiety per se or school refusal that is the pathogenic risk factor and (b) whether affected youngsters are specifically at risk of developing panic disorder rather than symptoms of general anxiety or phobias in later life. The present study of 74 adults who responded to media publicity found that a measure of early separation anxiety but not a history of school refusal was associated with risk of adult panic disorder according to DSM-III-R criteria. In contrast, separation anxiety scores were not associated with the presence or absence of general anxiety symptoms or phobic-avoidance in adulthood. Subjects with higher separation anxiety scores were more likely to have either a sibling or child with school refusal. Although the present study is limited in its method to mailed survey responses and, in part, to retrospective data, the results do provide additional support for Klein's influential separation anxiety theory of panic disorder.

Adult↗
PubMed1987

The safety of and the strain on the donor during thrombocytapheresis on the cell separator. A psychological study.

The study deals with the question as to what extent and under which existing personal characteristics does thrombocytapheresis on the cell separator (Fenwal CS-3000) lead to psychological stress for the blood donor? A comparison is made with the stress experienced during the established 450 ml full-blood donation. Because of the different procedural conditions, it was postulated that the donation on the cell separator creates greater psychological stress than the established type of donation. To examine this hypothesis, a sample group of 76 cell-separator and full-blood donors were asked to complete a questionnaire concerning the stress they experienced and their attitude before, during, and after the donation. Their personalities were also examined. A control group of 45 full-blood donors was examined in the same way. No significant statistical difference was found regarding the extent of stress under both conditions of donation. These findings are verified by the general satisfaction expressed by the cell-separator donors. Although it is confirmed that the personal cost to the donor is greater, it involves, on the other hand, a greater commitment, a positive evaluation of the more intimate involvement of the donor, and an increase in self-esteem. Since it could not be proven that there is an increase in the amount of stress during cell-separator donation, it also seems plausible that no clear decrease in stress would be observed after repeated donations. On the whole, it seems that the cell separator is well accepted by the donors. The extent of experienced stress is related to the donor's habitual anxiety and negative expectations. Since negative expectations can be influenced, it is to be expected that an informative conversation in a quiet atmosphere would further decrease the stress experienced.

Adaptation, Psychological↗
PubMed1997

Protein tyrosine phosphatases mediate cell readhesion in alveolar epithelial cells mechanically separated from in vitro matrix.

Alveolar epithelial type II cells are the progenitor cells for restoring the alveolar epithelial barrier after acute lung injury. During repair of lung injury, the alveolar epithelial type II cells reepithelialize denuded air spaces, a process that involves breaking and reforming cell adhesions. A novel technique of mechanical separation of cultured alveolar epithelial cells from in vitro matrix was used to examine the intracellular signals that result when alveolar epithelial cell adhesions are broken. The results show that the tyrosine phosphorylation levels of focal adhesion kinase, paxillin, and pp60(src) decreased immediately after mechanical separation of the cells. Levels returned to nearly normal by 24 h after mechanical separation. Paxillin and pp60(scr) coprecipitated with focal adhesion kinase regardless of their phosphorylation state. Interestingly, the tyrosine phosphorylation level of the mitogen-activated protein kinase, p42(erk2), increased 15 min after mechanical separation. Preincubation of cell monolayers with phenylarsine oxide, a protein tyrosine phosphatase inhibitor, blocked the decrease in tyrosine phosphorylation levels of focal adhesion kinase, paxillin and pp60(src). Phenylarsine oxide incubation also prevented readhesion of mechanically separated cells at 24 h, but genistein, a tyrosine kinase inhibitor, had no effect. We conclude that protein tyrosine phosphatases are activated immediately after cultured alveolar epithelial cells are mechanically separated from in vitro matrix, and their activation is required for alveolar epithelial cell readhesion.

Animals↗
PubMed1993

Separation anxiety in panic disorder.

OBJECTIVE: A number of researchers have provided evidence that separation anxiety is an important antecedent or current affect in panic disorder. The objective of this pilot study was to test this hypothesis by comparing dreams, screen memories, and life situations of panic disorder patients with those of comparison patients. METHOD: A recent dream with associations, screen memories with associations, and life situations at onset of symptoms were recorded verbatim during semistructured interviews with 20 patients with DSM-III-R panic disorder and 20 comparison subjects upon referral to a private outpatient practice. A judge blind to the diagnoses rated each of the dreams, screen memories, and life situations separately on each of the 10 Gottschalk-Gleser Content Analysis Scales, thereby measuring manifest and latent death, mutilation, separation, guilt, and shame anxiety and overt and covert hostility in each group. RESULTS: mean separation anxiety scores were significantly higher in both the dreams and screen memories of the panic disorder patients than in the comparison patients. Mean scores for covert hostility directed outward were significantly higher in the dreams of the panic disorder patients than in the comparison patients. CONCLUSIONS: The hypothesis that separation anxiety is a prevalent affective undercurrent in the dreams and screen memories of panic disorder patients was supported. Significantly higher covert hostility in the panic disorder patients' dreams may support Bowlby's observation that people with high separation anxiety tend to disavow their anger.

Anxiety, Separation↗
PubMed1976

Studies of cellular differentiation by automated cell separation. Two model systems: Friend virus-transformed cells and Hydra attenuata.

The automated high-speed analysis and separation of cells on the basis of spectroscopic parameters has been applied to studies of cellular differentiation in two systems. The temporal changes following induction of differentiation by dimethylsulfoxide in the Friend virus-transformed erythroid cells were quantitated by multiparameter analysis leading to the separation of discrete subpopulations. Thus, following induction, cell size decreased as measured by light scattering, the number of H-2 histocompatibility antigen sites decreased as measured by indirect fluorescent antibody binding, the number of lectin-binding sites per cell increased as measured by fluorescein-labeled concanavalin-A and the microviscosity of the hydrocarbon region of the plasma membrane increased as determined by the fluorescence emission anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene. Cells were separated on the basis of several of these parameters and analyzed for their hemogloglobin content by benzidine staining. Examination of cells separated according to the anisotropy parameter showed that high anisotropy values were correlated with (a) small cell size, (b) positive staining with benzidine and (c) pronounced reactivity with fluorescent antibody to the erythrocyte protein spectrin. Disaggregated cells from Hydra attenuata were selectively stained with the dyes rhodanile blue, 7-(p-methoxybenzylamino)-4-nitrobenz-2-oxa-1,3-diazole and fluorescamine. Distribution analyses and preliminary separations indicated the feasibility of obtaining homogeneous classes of cell types in a viable state. The experiments with emission anisotropy represent the first analyses and separations of single cells on the basis of fluorescence polarization. Many other uses of this technique are anticipated.

Autoanalysis↗
PubMed2005

Particle separation using ultrasound can be used with human shed mediastinal blood.

BACKGROUND: Shed mediastinal blood collected by cardiotomy suction has been shown to be a large contributor to lipid microemboli ending up in different organs. The aim of this study was to test the separation efficiency on human shed blood of a new separation method developed to meet this demand. METHODS: Shed mediastinal blood collected from the pericardial cavity of 13 patients undergoing cardiac surgery with cardiopulmonary bypass was collected. The blood was processed in an eight-channel parallel PARSUS separator, and separation efficiency was determined. RESULTS: Erythrocyte recovery, in terms of a separation ratio, varied between 68% and 91%. Minor electrolyte changes took place, where levels of sodium increased and levels of potassium and calcium decreased. CONCLUSION: This study demonstrates that PARSUS technology can be used on human shed mediastinal blood with good separation efficiency. The technology is, thereby, suggested to have future clinical relevance.

Blood Transfusion, Autologous↗
PubMed1992

An evaluation of cell separation techniques in a model mixed cell population.

Muscle precursor cells may act not only as a means of inserting normal genes into diseased muscle fibres, in order to correct or alleviate a genetically inherited myopathy, but recent demonstrations have shown they may prove an invaluable tool for the expression of, and systemic dissemination of, non-muscle gene products. If muscle precursor cells are proved to act as such widespread vectors in terms of gene therapy, then it is imperative that methods are properly elucidated to produce large populations of pure viable myogenic cells for such purposes. In the past, many methods of cell separation have been investigated but carry with them the problems of either a lack of myogenic purity of the population or poor percentage recovery of the original cell population. In the present work we have investigated two methods for segregating myogenic from non-myogenic cells and have critically reviewed the efficiency of separation of the two techniques used. To obtain a quantitative measure of separation efficiency, segregation was carried out on a 1:1 mixture of murine C2 myogenic and murine 3T3 fibroblastic cells. To distinguish between C2 and 3T3 cells, the latter were prelabelled with the fluorescent strain carboxyfluorescein diacetate succinimyl ester (CFSE). Once incorporated into the cell, CFSE remains there, thus preventing transfer of the label to C2 cells. Both methods of separation used depend on the affinity of myogenic cells for the monoclonal antibody Mab H28, which specifically binds to the mouse neuronal cell adhesion molecule N-CAM, but differ in that one method, "panning", completes segregation by adherence of N-CAM positive cells to a dish precoated with secondary IgG antibody whereas in the other separation proceeds by the use of commercially available IgG-coated magnetic beads. Results indicate magnetic bead separation to be more efficient than panning if the beads are precoated with 0.1% gelatin.

3T3 Cells↗
PubMed1992

[Investigation of a new method for separation of neutrophils from a small volume of human blood].

We have already reported a neutrophil separation method for the multiple simultaneous measurement of neutrophil chemiluminescence. However, when the reported separation method was used, at least 4 ml of venous blood was needed to collect enough neutrophils for chemiluminescent measurement. Because of this blood volume, there is a limitation on applications of the multiple simultaneous method for neonates, infants, and in some clinical situations. To expand the application of this neutrophil chemiluminescence measurement into clinical and health science areas, we have developed a new method for separation of neutrophils from a relatively small amount of blood (500 microliters). In addition, the influences of remaining red blood cells and hemoglobin levels in the neutrophil fraction on the chemiluminescence were examined to determine the necessity for elimination procedures. The new separation method used a capillary tube (length, 130 mm; outside diameter, 5 mm; thickness, 0.8 mm) with density gradient reagents (Histopaque 1077 and 1119). After centrifugal separation (500 g, 30 min), the neutrophil fraction was isolated with 93.1 +/- 4.7% purity and 60.6 +/- 11.1% yield. This purity and yield were comparable to or better than those with the previously reported method, while levels of remaining red blood cells and hemoglobin were about the same. Remaining red blood cells and hemoglobin in the neutrophil fraction acted on the chemiluminescence as a quencher. For the correct estimation of neutrophil chemiluminescence, elimination of remaining red blood cells and hemoglobin in the neutrophil fraction is necessary. This new neutrophil separation method is a very useful method, especially for cases in which available blood amounts are limited.

Cell Separation↗
PubMed1999

The nature of the mother's tie to her infant: maternal bonding under conditions of proximity, separation, and potential loss.

Attachment has generally been examined from the infant's perspective. We focused on mothers' post-partum thoughts and behaviors. Guided by an ethological approach, maternal bonding was examined under conditions of proximity, separation, and potential loss. Ninety-one mothers were interviewed: mothers of full-term infants who maintained continuous proximity to the infant, mothers of healthy premature infants who were separated from the infant, and mothers of very low birthweight infants who experienced potential loss and prolonged separation. Mothers of term infants reported medium-to-high levels of preoccupations with thoughts of infant safety and well-being. Preoccupations increased with separation (Group 2) and significantly decreased with impending loss (Group 3). Attachment behaviors and representations were the highest among mothers of term infants and declined linearly with the duration of mother-infant separation. Maternal trait anxiety and depression were related respectively to higher levels of preoccupations and reduced attachment behaviors and representations, independent of the infant medical condition and mother-child separation. Discussion focused on the comparability of maternal and infant attachment in relation to the neurobiological system underlying bond formation.

Adult↗
PubMed1975

[Separation of reb blood cells in the isopyknic dextran and albumin density gradient].

The separation of red blood cells according to their maturity and age was compared in the isopyknic dextran and albumin density gradients, and the quality of separation was appraised by selected criteria (density distribution curves, accumulation of reticulocytes, activity of G-6-PD). The achieved quality of separation, as measured by the quotient of G-6-PD activity in the cells of lower density/G-6-PD activity in cells of higher density is of equal value in the two media. In the dextran gradient, separation of old cells seems to be accomplished more readily, while albumin affords better separation of young erythrocytes. The reticulocytes are in both media 4-5 mg/ml lighter than the total cell population. When shifting pH from 7.4 to 6.8 both in erythrocytes and reticulocytes the density decreases by 3-6 mg/ml irrespective of the type and pretreatment of the separation medium.

Albumins↗
PubMed1975

Separation of human lymphoid cells by preparative cell electrophoresis.

By means of free-flow electrophoresis a preparative separation of cells according to their surface charge is achieved. The method has been applied to human lymphocytes of blood and of tonsils. Technical details are described and discussed with respect to optimal separation conditions, reproducibility of results and viability of the separated cells. Unseparated cells, pooled separated cells and cells corresponding to different electrophoretic mobilities are compared for stimulation in response to PHA, determination of cells bearing membrane immunoglobulins, colony growth of hemopoietic stem cells and DNA syntheses in vitro. It is concluded that reproducible results can be obtained, that the biological activities of the cells after separation are unaltered and that a separation or at least an enrichment of cells with certain properties can be achieved.

Adenoids↗
PubMed1992

Infant gender and sibling dyad influences on maternal separation anxiety.

The purpose of this longitudinal study was to compare maternal separation anxiety by gender of infants and sibling dyad in 40 employed, second-time mothers. Subjects completed two parallel measures of separation anxiety, the Maternal Separation Anxiety. Scale (MSAS) and the Interview-Based Rating Scales (IBRS). The mothers completed the MSAS when the first infant was born and again 2 years later when their second infant was 7 months old (mean age). Following completion of the MSAS (for the second child), the mothers were interviewed and rated on their separation concerns (IBRS) related to both children. No gender differences were noted when t-tests were computed on mothers' MSAS mean scores, but the IBRS revealed that mothers of second-born sons were significantly more anxious about separation (t = 2.01, p < .05) than were mothers of second-born daughters. One-way General Linear Model comparisons of both measures, for the four sibling dyads, revealed significant differences related to the dyad composition. Mothers of girl/girl dyads were significantly more anxious about separation as measured by the MSAS. On the IBRS, mothers of boy/boy dyads were significantly more anxious and mothers of boy/girl dyads were significantly less anxious when compared to mothers of the other dyads.

Adult↗