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PubMed1997

The function of neutrophils isolated by a magnetic antibody cell separation technique is not altered in comparison to a density gradient centrifugation method.

Most comparative studies on neutrophil (PMN) isolation techniques have shown either activation or functional impairment of the cells due to the different separation processes. We have established a preparation method for PMN from human whole blood employing iron tagged, magnetizable antibodies against the cell surface antigen CD15. The aim of our study was to test whether this magnetic separation (MACS) alters cellular functions of PMN in comparison to a conventional density gradient technique (Percoll). The purity, cell yield, and pre-activation of the cells were evaluated. The latter was assessed by quantifying the expression of the integrin CD11b using flow cytometry. Furthermore, as functional tests, cell morphology and the oxidative burst reaction were investigated. We have shown that the use of 'magnetic' antibodies leads to highly purified PMN (> 99% of isolated leukocytes), while there is still contamination by eosinophils (about 6%) after Percoll separation. Platelet contamination was about the same in both procedures (approx. one platelet per two PMN). The basal expression of CD11b and, hence, neutrophil activation, was significantly lower and the upregulation of CD11b in response to FMLP was more pronounced after magnetic separation, as compared to density gradient centrifugation. The MACS technique did not lead to polarisation of PMN, nor did it affect the oxidative burst. This study suggests that magnetic separation is a simple, time-saving technique, yielding highly purified and functionally intact PMN.

Centrifugation, Density Gradient↗
PubMed2001

Parent-child concordance for separation anxiety: a clinical study.

There is tentative evidence supporting a familial basis for separation anxiety. The present study aimed to examine parent-child concordance for that subtype of anxiety. Fifty-four children diagnosed with anxiety disorders and their parents (54 mothers and 29 fathers) were recruited from two juvenile anxiety clinics. Sixty-three percent of children diagnosed with juvenile separation anxiety disorder had at least one parent who suffered from the putative adult variant of the disorder (odds ratio = 11.1) (P < 0.001). Affected parents reported high levels of separation anxiety in their own childhoods. Juvenile separation anxiety disorder in children was not associated with any other parental diagnosis. The small sample size and other potential biases caution against definitive conclusions being drawn, but the present data add to existing evidence that separation anxiety may aggregate in families.

Adult↗
PubMedDate not supplied

Continuities of separation anxiety from early life into adulthood.

The study investigates whether a putative diagnosis of separation anxiety disorder can be identified in adulthood and whether there are continuities between juvenile and adult forms of the disorder. Seventy patients with conventional adult diagnoses of panic disorder and generalized anxiety disorder attending an anxiety clinic were administered an interview and checklist to assess separation anxiety (SA) symptoms in adulthood. Memories of early SA were assessed using the Separation Anxiety Symptom Inventory (SASI). A subsample (n = 31) was used to calibrate the checklist against assignment to a category of adult separation anxiety disorder (ASAD) based on the structured interview. In an expanded sample (n = 70), patients assigned to the ASAD category returned statistically higher scores on the SASI, with the severity of juvenile SA symptoms accounting for 33% of the variance of adult SA scores (p < .001). Assignment of subjects to the putative ASAD category was not associated with any conventional adult anxiety diagnosis and symptoms of SA appeared to predate the onset of the other anxiety disorders. One possible explanation for the data is that, in some individuals, early onset separation anxiety disorder may persist into adulthood, but the symptoms may either be overlooked or, alternatively, obscured by secondary features such as panic.

Adolescent↗
PubMed2001

Obsessive-compulsive disorder and separation anxiety co-morbidity in early onset panic disorder.

BACKGROUND: This study was undertaken to examine the relationship between anxiety co-morbidity and age of onset of panic disorder. METHODS: Age of onset of panic disorder and co-morbid anxiety disorders were assessed among 201 panic disorder probands with childhood separation anxiety disorder, obsessive-compulsive disorder, obsessive-compulsive symptoms, social phobia and specific phobia as part of a clinician-administered lifetime diagnostic interview. A generalized linear model was used to test the association between each anxiety co-morbidity and age of panic disorder onset while simultaneously controlling for the potential confounding effects of sociodemographic characteristics and other psychiatric co-morbidity. RESULTS: Earlier onset of panic disorder was found in patients with co-morbid obsessive-compulsive disorder, obsessive-compulsive symptoms and separation anxiety disorder, but not simple phobia or social phobia. Patients with both childhood separation anxiety disorder and obsessive-compulsive disorder had an even earlier panic onset than those with either childhood separation anxiety disorder or obsessive-compulsive disorder. CONCLUSIONS: The association between anxiety co-morbidity and earlier onset of panic disorder is specific to obsessive-compulsive disorder and childhood separation anxiety disorder.

Adolescent↗
PubMed2004

On-chip free-flow magnetophoresis: continuous flow separation of magnetic particles and agglomerates.

The separation of magnetic microparticles was achieved by on-chip free-flow magnetophoresis. In continuous flow, magnetic particles were deflected from the direction of laminar flow by a perpendicular magnetic field depending on their magnetic susceptibility and size and on the flow rate. Magnetic particles could thus be separated from each other and from nonmagnetic materials. Magnetic and nonmagnetic particles were introduced into a microfluidic separation chamber, and their deflection was studied under the microscope. The magnetic particles were 2.0 and 4.5 microm in diameter with magnetic susceptibilities of 1.12 x 10(-4) and 1.6 x 10(-4) m(3) kg(-1), respectively. The 4.5-microm particles with the larger susceptibility were deflected further from the direction of laminar flow than the 2.0-microm magnetic particles. Nonmagnetic 6-microm polystyrene beads, however, were not deflected at all. Furthermore, agglomerates of magnetic particles were found to be deflected to a larger extent than single magnetic particles. The applied flow rate and the strength and gradient of the applied magnetic field were the key parameters in controlling the deflection. This separation method has a wide applicability since magnetic particles are commonly used in bioanalysis as a solid support material for antigens, antibodies, DNA, and even cells. Free-flow magnetophoretic separations could be hyphenated with other microfluidic devices for reaction and analysis steps to form a micro total analysis system.

Antibodies↗
PubMed1999

Cell separation on microfabricated electrodes using dielectrophoretic/gravitational field-flow fractionation.

Dielectrophoretic/gravitational field-flow fractionation (DEP/G-FFF) was used to separate cultured human breast cancer MDA-435 cells from normal blood cells mixed together in a sucrose/dextrose medium. An array of microfabricated, interdigitated electrodes of 50 microns widths and spacings, and lining the bottom surface of a thin chamber (0.42 mm H x 25 mm W x 300 mm L), was used to generate DEP forces that levitated the cells. A 10-microL cell mixture sample containing approximately 50,000 cells was introduced into the chamber, and cancerous and normal blood cells were levitated to different heights according to the balance of DEP and gravitational forces. The cells at different heights were transported at different velocities under the influence of a parabolic flow profile that was established in the chamber and were thereby separated. Separation performance depended on the frequency and voltage of the applied DEP field and the fluid-flow rate. It took as little as 5 min to achieve cell separation. An analysis of the DEP/G-FFF results revealed that the separation exploited the difference in dielectric and density properties between cell populations. The DEP/G-FFF technique is potentially applicable to many biological and biomedical problems, especially those related to microfluidic systems.

Algorithms↗
PubMed2002

Separation of human lymphocytes from citrated blood by density gradient (NycoPrep) centrifugation: monocyte depletion depending upon activation of membrane potassium channels.

Routine one-step centrifugation procedures (Lymphoprep = LP, Percoll) commonly used for separation of blood cells split the cells into two major fractions. After centrifugation the mononuclear cells (MNC = monocytes and lymphocytes) are located on the top of the separation fluid, whereas erythrocytes and granulocytes have sedimented to the bottom. We now show that a relatively pure lymphocyte suspension can be obtained by one-step centrifugation of citrated blood by using NycoPrep (NP = iohexol), a nonionic X-ray contrast agent. With this gradient medium also the monocytes pass to the bottom, leaving lymphocytes on the top. In parallel separations with LP, which contains Ficoll and a fully dissociated sodium salt of a contrast medium, the results were as usual, i.e. approximately 70-85% lymphocytes and 30-15% monocytes in the top fraction. The monocyte depletion with NP depended upon the use of citrated (ACD) blood and a proper balance of density and osmolality of the gradient medium, and was enhanced by 20 min preincubation with CaCl2 at room temperature. Monocyte depletion could not be obtained with LP. Under optimal conditions (density 1.075 g/ml, osmolality 280-300 mOsm/kg), the monocyte admixture amounted to approximately 1 (0-2)%, in separations with buffy coat samples. For freshly drawn blood, it was necessary to slightly modify the NP solution. The monocyte depletion was counteracted by blockers of K+ channels or by KCl in the cell suspension. Following incubation in NP of Percoll-separated cells, an enhanced release of K+ was observed. The results are interpreted as follows: NP mediates the opening of K+ channels of MNC, which leads to efflux of K+, accompanied with associated anions (Cl-). This reduces the osmolality inside the cells which therefore expel water to maintain osmotic equilibrium. In this regard it appears that monocytes are more sensitive than lymphocytes, their density therefore increasing more, so that they are able to pass the density barrier otherwise exerted by the gradient medium.

Blood Platelets↗
PubMedDate not supplied

Density gradient separation of peripheral blood stem cells: comparison of an automated cell processing device and manual methods.

Peripheral blood stem cells were collected from normal donors by leukapheresis on a cell separator. The leukapheresis product contained 1.5 x 10(10) mononuclear cells (MNCs) and was divided into two aliquots that underwent either automated or manual density gradient separation with ficoll-hypaque and subsequent washing. In the automated process, recovery of MNCs was 85 percent, reduction in platelet content was 64 percent, and the final hematocrit (Hct) was less than 1 percent. The manual separation resulted in 76-percent MNC recovery, a 79-percent reduction in platelet content, and a final Hct of less than 1 percent. The purified MNCs were then placed in methylcellulose culture at a concentration of 4 x 10(5) MNCs per mL. Quadruplicate 1-mL aliquots were cultured, and colonies were counted and classified on Day 14. Comparison of automated and manual ficoll-hypaque separations demonstrated no differences in the total, erythroid, or granulocyte-macrophage colony numbers. The cell processor used is fast, reliable, uncomplicated, and provides a sterile product containing progenitor cells that are not adversely affected by the automated ficoll-hypaque separation.

Cell Separation↗
PubMed1999

Plateletpheresis efficiency: a comparison of the spectra LRS and AMICUS separators.

BACKGROUND: Optimizing the yields of plateletpheresis by increasing collection efficiencies may provide several benefits: to patients, by increasing the dose in single-donor platelet (SDP) units; to the collection center, by increasing the percentage of components that may be split into double units; and/or to the donor, by reducing the duration of donation. STUDY DESIGN AND METHODS: A prospective, randomized study was undertaken to compare the efficiency of platelet collection in paired donations by 21 donors on two cell separators (Spectra LRS, version [v] 5.1; and AMICUS, v2.37). The order of donation was randomly assigned; donations were performed at least 2 weeks apart. A fixed blood volume (4000 mL) was processed by utilizing standard protocols. Findings were confirmed in a retrospective, matched study that compared the two separators by using fixed collection times (90 min). RESULTS: The AMICUS and Spectra LRS separators consistently produced white cell-reduced (<1 x 10(6) white cells) components. The AMICUS harvested 32 percent more platelets on average (median, 4.9 x 10(11); range, 1.5-8.7 x 10(11)) than the Spectra LRS (median, 3.7 x 10(11); range, 2.1-7.7 x 10(11)) (p = 0.03), with mean times of 71.5 and 66.0 minutes (p = 0.03), respectively, to process 4000 mL. Mild donor reactions tended to be more common on the AMICUS separator, which used significantly more ACD (median 482 mL vs. 389 mL; p<0.0001) than on the Spectra LRS. CONCLUSIONS: The AMICUS separator harvested more platelets per unit of blood volume processed than the Spectra LRS. Possible benefits include increased dose in each single-donor unit, increased double-unit harvests, and/or shorter donation time.

Cell Separation↗
PubMedDate not supplied

Separation of sperm through a 12-layer percoll column decreases the percentage of sperm staining with quinacrine.

Previous methods of enriching sperm with a higher percentage of Y-bearing sperm have been questioned because the claims that Y enrichment was present were based on quinacrine staining of the Y chromosome, and the enrichment was not confirmed by polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) techniques. A technique was evaluated that theoretically could increase the percentage of X-bearing sperm by isolating a fraction of the "heaviest" sperm by passing them through 12 layers of discontinuous Percoll gradient. Initially 12 specimens were checked both before and then after separation with 12 layers of Percoll for percentage of Y sperm. The median for baseline Y percentage was 49% and after processing the percentage of Y dropped to 10%. An additional 19 specimens were checked after separation only. The median was 19%. The sample with the lowest preseparation % of quinacrine staining sperm was 45% and the highest was 54%. After 12-layer Percoll, the lowest percentage was 3% and the highest was 24%. There have been claims that quinacrine staining can falsely increase apparent Y-bearing sperm enrichment following certain separation procedures, e.g.. albumin separation, by nonspecific staining of autosomal chromosomes. If anything, then, it should falsely decrease X-bearing sperm enrichment. Thus, 12-layer Percoll separation may actually enrich for X-bearing sperm or possibly this procedure somehow nonspecifically inhibits the ability of quinacrine to stain the Y chromosome.

Cell Separation↗
PubMed2002

Hypothalamic-pituitary-adrenal responses to weight loss in mice following diet restriction, activity or separation stress: effects of tyrosine.

We have studied three different types of weight-loss stress caused by Diet restriction, Activity or Separation, for their effects on the hypothalamic-pituitary axis in young female mice and their responses to tyrosine 100 mg/kg/day. Plasma was assayed for ACTH and glucocorticoid determinations, and brain catecholamine concentrations were measured by HPLC/ECD. A similar weight loss of 24-28% was observed in the models despite significant differences in food intake. Diet restriction to 60% and Separation models produced a significant increase in hypothalamic noradrenaline (p < 0.01), while there was a significant decrease (p < 0.05) in the Diet restriction to 40% that was restored after tyrosine. After Activity, noradrenaline levels did not change. ACTH concentrations decreased following Diet restriction (p < 0.05) but were unaffected by Separation or Activity. The peripheral glucocorticoid response increased significantly after Activity and Diet restriction (p < 0.001), but decreased significantly after Separation (p < 0.001). Tyrosine increased glucocorticoid concentrations in the Activity and Separation models (p < 0.05), but not after Diet restriction. Despite similar weight loss in the three models there were no predictable associations between hypothalamic noradrenaline metabolism and plasma ACTH or glucocorticoid concentrations. Tyrosine might alleviate some of the different pathophysiological problems associated with the stress of weight loss.

Adrenocorticotropic Hormone↗
PubMed2004

Relations between young children's responses to the depiction of separation and pain experiences.

This study examined relations between young children's representations of separation and pain experiences in 60, 4- and 5-year-old children. Separation representations were assessed with the Separation Anxiety Test (SAT) and pain representations were assessed by examining responses to pictures of children about to experience pain in the presence of parent figures. Results showed that representations of separation and pain experience were systematically related and the patterns were not accounted for by the child's ability to differentiate emotional states, language ability, or reports of emotional regulation. These findings are consistent with Bowlby's (1982) concept of secure base behaviour in response to a variety of distress, and support the hypothetical construct of an internal working model of attachment which organizes children's behaviours, thoughts, and feelings in response to both separation experience and painful events.

Adult↗
PubMed1974

Studies on dispersed pancreatic exocrine cells. II. Functional characteristics of separated cells.

The functional characteristics of separated guinea pig pancreatic exocrine cells have been examined following dissociation of the gland by a procedure described in the previous paper (J. Cell Biol. 1974. 63:1037). The ability of isolated cells to incorporate labeled amino acids into secretory proteins was assessed biochemically and by quantitative electron microscope autoradiography. Incorporation remained linear for up to 4-h incubation at levels equivalent to those of pancreatic slices; over 95% of the exocrine cells in the population were viable, and all appeared to be equally active in incorporating amino acids. The capacity of separated cells to transport, concentrate, and store exportable proteins was monitored by electron microscope autoradiography on populations pulse labeled with [(3)H]leucine and chase incubated for 4 h. The same overall pathway previously mapped in pancreatic slices was followed by secretory proteins in separated cells although in quantitative studies a defect was noted in the rate of conversion of condensing vacuoles to zymogen granules. Secretogogue responsiveness was assessed by monitoring discharge of labeled secretory proteins or of amylase in response to carbamylcholine and caerulein to the medium. While the separated cells released secretory proteins linearly for up to 4 h in response to both secretogogues, the net release was approximately 50% less than previously noted for pancreatic slices and required a ten times higher concentration of stimulant. The defect may represent alteration in receptors due to the protease used for dissociation. Our data indicate, however, that separated exocrine cells retain their ability to process secretory proteins stepwise and vectorially which is consistent with preservation of structural polarity.

Amylases↗
PubMed1997

Efficiency of sex pre-selection of spermatozoa by albumin separation method evaluated by double-labelled fluorescence in-situ hybridization.

To evaluate the separation efficiency of Ericsson's two- and three-layer albumin separation methods, semen samples from 21 healthy males were studied. Seven patients already had two or more sons, another seven had two or more daughters and the other seven had primary infertility due to female factors. The semen samples were divided into three aliquots: one remained unprocessed initially, the other two aliquots went through two- and three-layer albumin separation methods respectively. All samples were then stained with X-Y double staining probes. In each group, four or five samples were processed at room temperature, and two or three at body temperature (37 degrees C). The labelling efficiency of X-Y double staining probe was over 99%. The X:Y sperm ratios were even in the original samples. The ratios of the X and Y spermatozoa were altered slightly but significantly after the two-layer (P < or = 0.05) or the three-layer (P < or = 0.005) separation. The alterations occurred only at room temperature. The X spermatozoa increased and the Y spermatozoa decreased, both to a small degree of difference (1.4-3.5%). Double fluorescence in-situ hybridization analysis therefore showed that albumin separation methods do not enrich Y spermatozoa.

Albumins↗
PubMed1998

Experiences in Hong Kong with the theory and practice of the albumin column method of sperm separation for sex selection.

Controversy still surrounds the human serum albumin (HSA) method for separation of X- and Y-bearing human spermatozoa. There is doubt about whether the procedure does enrich sperm samples for the chosen sex chromosome. We have applied the HSA separation method in a clinic in Hong Kong, using the method as described by Ericsson et al. [Nature, 246, 421-424 (1973)] taking care to keep the sperm recovery to <5% of the initial number. Aliquots of separated spermatozoa were examined for X- and Y-bearing spermatozoa by fluorescent in-situ hybridization (FISH) using appropriate DNA probes. Of 18 couples wanting boys, 13 had single boys, one had twin boys, and one had twins comprising one boy and one girl. Only three single girls were born. This success rate of 83% is significantly different (P < 0.001) from the usual expected ratio. There were four miscarriages, one in the third and one in the fourth week of pregnancy. The times of the others are not definitely known, but are thought to have occurred early in pregnancy. We lack information on three couples. The FISH procedure showed no change in the normal and equal numbers of X- and Y-bearing spermatozoa after the HSA separation procedure. This study confirmed that the HSA sperm separation method can bias the number of babies in favour of males. However, the theory that it does so by enriching the sperm samples with Y-bearing spermatozoa appears to be incorrect and some other theory has to be postulated. It is tentatively proposed that passage through the HSA inactivates X-bearing spermatozoa more than Y-bearing spermatozoa, even though this is not apparent simply on inspection of sperm motility.

Cell Separation↗
PubMed1994

Effects of maternal hospitalization in early childhood: anticipated anxiety associated with an analog separation for childbirth and surgery.

Assessed early childhood anticipation of anxiety in response to analog 3-day maternal hospitalization for surgery or childbirth. Reaction to analog nonhospital separation was also assessed. Within each condition, gender effects were examined. Participants were 120 first-grade children at four elementary schools. Between-groups comparisons showed maternal separation for surgery was anticipated as more anxiety provoking, whereas anxiety associated with separation for childbirth and nonhospital separation was anticipated as similar. In general, within each separation situation, girls and boys anticipated similar levels of anxiety.

Anxiety, Separation↗
PubMed1996

Traits of separation anxiety in boys with gender identity disorder.

OBJECTIVE: To assess the presence of traits of separation anxiety disorder in boys referred clinically for gender identity disorder. METHOD: One hundred fifteen boys were referred to a specialty clinic for concerns about their gender identity development. They were divided into two subgroups: one group met the complete diagnostic criteria for gender identity disorder; the other group did not meet the complete diagnostic criteria. The mothers of the boys were administered a structured interview schedule pertaining to separation anxiety disorder according to DSM-III criteria. RESULTS: A conservative definition of separation anxiety disorder showed no significant association with gender identity disorder; however, a liberal definition of separation anxiety disorder showed that it occurred significantly more often in the subgroup of boys who met the complete criteria for gender identity disorder than in the subgroup who did not meet the complete criteria (64.4% versus 38.1%, respectively). CONCLUSIONS: Boys with gender identity disorder show a high rate of co-occurring traits of separation anxiety. Reasons for this linkage require additional empirical study.

Anxiety, Separation↗
PubMed1988

Early separation anxiety and adult agoraphobia.

The authors compared 137 adult patients who had agoraphobia with 81 patients who had either simple or social phobia for a history of childhood and adolescent separation anxiety. Female agoraphobics reported significantly more childhood separation anxiety than female combined simple and social phobics; males showed no significant difference between diagnostic groups. The reported prevalence of separation anxiety in adolescence was relatively low, but agoraphobics of both sexes reported significantly more separation anxiety than combined simple and social phobics. There were no significant differences between groups for parental losses or severe family disruption during childhood. The results suggest that childhood separation anxiety is a risk factor in females but not in males for the later development of agoraphobia.

Adolescent↗