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PubMed1996

Advantages of using a cell separator and metrizamide gradients for human islet purification.

Human islet transplantation has a high rate of failure, often due to primary nonfunction, which suggests that islets are damaged during the processing of the pancreas. The preparation of human islets for transplantation is still a complex process that requires large teams of surgical and laboratory personnel. To overcome this problem, we have adopted the use of the IBM 2991 COBE cell separator and a metrizamide/Ficoll density medium that is easy to prepare. Twenty-seven pancreatic glands have been processed using the COBE cell separator, 23 of which were purified in metrizamide/Ficoll gradients and 4 in bovine serum albumin gradients. The results show an improvement of recovery and viability in these preparations when compared retrospectively with manual gradients. More importantly, the time required for purification was shortened to one fourth the usual time and total processing time is about half as long. Moreover, a team of two laboratory staff was regularly able to prepare islets for transplantation, reducing the separation time from 7 hr to 3.5 hr. We conclude that the automatic cell separator and metrizamide-based separation medium are useful modifications of current islet purification methods.

Adolescent↗
PubMed1984

Separation of diploid from aneuploid cells in acute lymphoblastic leukaemia.

A new method for the separation of diploid and aneuploid cells from blood of patients with leukaemia is described. Density gradient separation using a modified silica particle gradient was combined with analysis of cellular DNA content by flow cytometry. Submicroscopic levels of diploid or aneuploid cells were enriched up to 90% purity. Three patients with acute lymphoblastic leukaemia and no evidence of aneuploid cells after separation are in continuing complete remission after 24.5-47 months. Two patients with aneuploid cells detected after separation expired in relapse 0.5 and 3 months later. Cell separation prior to flow cytometric analysis might be a useful tool to detect submicroscopic levels of aneuploid leukaemic cells in blood and bone marrow, and to predict impending relapse.

Aneuploidy↗
PubMed1984

Plasma separation in patients with bronchial asthma, atopic dermatitis and hyperimmunoglobulinaemia E.

The effect of intensive plasma separation performed eight times within 5 weeks in four patients with atopic dermatitis, bronchial asthma and hyperimmunoglobulinaemia E was followed as regards clinical symptoms and changes in the concentrations of serum (S) IgE, S IgG, S IgA, S IgM, plasma complement C3 split products, S transferrin, blood eosinophils, chemotaxis of neutrophil cells and histamine metabolites in urine in samples obtained consecutively during the period of observation. The occurrence of circulating immune complexes (IC) was analysed by a polyclonal rheumatoid factor (pRF) agglutination inhibition assay and an IgE IC specific technique. IgE IC were demonstrated in three of the patients prior to plasma separation, complexed IgE was 2-3% of the total concentration of S IgE. In one patient complexes were detected by the pRF agglutination inhibition assay, also. In the three patients with IgE IC, the complexes disappeared during treatment, but recurred in two of the patients shortly after the last plasma separation. Shortly after eight separations the S IgE was reduced in all patients to a mean level of 46% of the pre-exchange concentrations. During the following 3 weeks the relative increase of S IgE in three of the patients was similar to the values obtained for S IgG. Serum IgG was subnormal in all patients during the period of treatment. Increasing numbers of eosinophils were observed in three of the patients after the fifth separation procedure. The histamine metabolite 1,4-methylimidazoleacetic (1,4- MIAA ) in urine was increased in all patients, but no significant changes were observed during the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1981

Partial separation and biochemical characteristics of periportal and perivenous hepatocytes from rat liver.

Suspensions of enzymatically prepared hepatocytes from starved rats were separated according to their buoyant density at 12 degrees C in linear, isosmotic gradients of metrizamide, centrofuged at low speed for a relatively short time. The recovery of cell protein was 86%. Hepatocytes of high viability formed a single band around 1.10 g/cm3 and were recovered as four density populations (P1-P4) form low to high density, respectively. The content of protein was significantly lower in population P1, while the content of neutral fat or the averaged cell size was similar in the various populations. The specific activity of alanine aminotransferase increased in the order P1-P4. The distribution of this enzyme within the intact liver acinus obtained by others indicate that a partial separation of periportal and perivenous hepatocytes had occurred. The activity patterns of lactate dehydrogenase, glutamate dehydrogenase, isocitrate dehydrogenase (NADP+) and pyruvate kinase, also with known intra acinar distributions, supported this conclusion. The hepatocytes showed signs of shrinkage after separation, but since they retained a normal ultrastructure, most enzyme activities and viability, the present technique was regarded superior to previous procedures of hepatocyte separation by density. The degree of separation was calculated from an equation (see Appendix), and the periportal/perivenous ratio for parameters measured in density populations can be obtained. The specific activity of phosphofructokinase, alcohol dehydrogenase and aldehyde dehydrogenase showed no differences between populations. However, the ratio high-Km/low-Km aldehyde dehydrogenase increased in the order P4-P1.

Alcohol Oxidoreductases↗
PubMed1988

Molecular design of materials for cell separation.

There has been a strong demand in biomedical sciences to isolate viable cell populations with high yield and purity. An important facet of this work was to develop new polymeric adsorbent for the separation of lymphocyte subpopulations. Based on our strategy of separating cells through their differential ionic affinity toward multiphase-structured adsorbent with ionically derivatized microdomains, a series of poly(2-hydroxyethyl methacrylate)/polyamine graft copolymers (HA copolymers) was prepared. HA copolymer columns were found to show specific adsorption affinity toward B lymphocytes, and allows for separation of B and T lymphocytes in high yield and purity with a short operating time. Separation mechanism involved in the resolution of B and T lymphocytes by HA copolymer column is discussed in this paper. Further, photo-induced desorption of cells from the adsorbent derivatized with photo-responsive functional group (azobenzene group) was demonstrated to emphasize the feasibility of photo-regulated chromatography as a novel tool in cell separation technology.

B-Lymphocytes↗
PubMed2005

Computational simulation of a non-newtonian model of the blood separation process.

The aim of this work is to construct a computational fluid dynamics model capable of simulating the transient non-Newtonian process of apheresis. A Lagrangian-Eulerian model has been developed which tracks the blood particles within a two-dimensional flow configuration. Within the Eulerian method, the fluid mass and momentum conservation equations within the separator are solved using the density and the viscosity is calculated from the blood particle concentrations. Subsequently, the displacement of the blood particles is calculated with a Lagrangian method. Hawksley's model for the density of supensions is used in the variable density calculation. The viscosity is calculated with two models based on Vand's rigid particle suspension viscosity concepts, followed by the flow field calculation in the separator. Simulations were performed for various inlet hematocrit values and separator lengths. The simulations are in satisfactory agreement with experimental results reported in literature, indicating a complete separation of plasma and red blood cells (RBCs), as well as nearly complete separation of red blood cells and platelets. No hemolysis was observed in the simulations because the shear rate remained under the critical value of 150 N/m2.

Biophysical Phenomena↗
PubMed1995

Genetic factors in early separation anxiety: implications for the genesis of adult anxiety disorders.

An important contemporary conceptualization of anxiety has suggested that heightened early separation anxiety is specifically associated with the risk of adult panic disorder, with hereditary factors underlying that cluster of anxiety disorders. Yet there is a dearth of studies examining whether early separation anxiety is inherited. The present twin study, based on a retrospective approach, revealed a substantial genetic contribution to separation anxiety in females but not in males, with unique environmental influences being important in both gender groups. Although speculative, an evolutionary explanation is offered to account for the apparent gender difference in the inheritance of early separation anxiety. It is hypothesized that, in some women, phylogenetic vestiges of separation anxiety may conflict with their need to compete in an individualistic manner in the modern workplace. Whether such an attachment-autonomy conflict accounts for the increased rate of panic disorder and agoraphobia in women is worthy of further study.

Adolescent↗
PubMed1987

The effects of RPM and recycle on separation efficiency in a clinical blood cell centrifuge.

A COBE blood cell centrifuge, model 2997 with a single stage channel, was modified to allow computer controlled sampling, and to allow recycle of red blood cells (RBCs) and plasma streams using bovine whole blood. The effects of recycle of the packed RBC and plasma product streams, and of the centrifuge RPM on platelet and white blood cell (WBC) separation efficiencies were quantified using a central composite factorial experimental design. These data were then fit using second order models. Both the model for the WBC separation efficiency and the model for the platelet separation efficiency predict that RPM has the greatest effect on separation efficiency and that RBC and plasma recycle have detrimental effects at moderate to low RPM, but have negligible impact on separation efficiency at high RPM.

Animals↗
PubMed1997

[Trial for isolation of Helicobacter pylori form fecal specimen by immunomagnetic beads separation].

In order to clarify the possibility of feco-oral transmission of Helicobacter pylori, we have employed an immunomagnetic beads (IMB) separation technique and tried to isolate H. pylori from fecal samples. Microaerophilic cultivation and PCR of H. pylori were positive when H. pylori (10(3) cfu) was suspended in Hanks' balanced salt solution (HBSS) for the IMB separation technique. In contrast, when a lower number of H. pylori (10(2) cfu) was suspended in HBSS, 2nd PCR was positive but cultivation was negative. After IMB separation technique from fecal sample containing H. pylori (2 x 10(4) cfu), 20 cfu of H. pylori was recovered by culture. In this case, 1st PCR was negative but 2nd PCR was positive. From a fecal sample of a patient infected with H. pylori, H. pylori was detected only by combination of IMB separation and PCR, although H. pylori was not recovered by culture. These results indicate that IMB separation technique followed by cultivation or PCR would be useful for estimation of the presence of H. pylori fecal samples.

Enzyme-Linked Immunosorbent Assay↗
PubMed1998

Improvement of the immunomagnetic separation method selective for Escherichia coli O157 strains.

Immunomagnetic separation is a useful enrichment method selective for Escherichia coli O157 cells against non-O157 E. coli cells from a preenrichment culture. However, E. coli cells are adsorbed onto a solid surface nonspecifically. With the conventional immunomagnetic separation method, this nonspecific adsorption interfered with immunomagnetic separation. It was found that this interference could be reduced with a low-ionic-strength solution. When immunomagnetic separation was carried out with this solution, the proportion of E. coli O157 cells to non-O157 E. coli cells increased from 9.6 to 31.4 times compared to the proportion obtained by the conventional immunomagnetic separation method. The effectiveness of this solution was successfully evaluated by the use of E. coli O157-spiked samples.

Adsorption↗
PubMed1998

Effect of shipment, storage, anticoagulant, and cell separation on lymphocyte proliferation assays for human immunodeficiency virus-infected patients.

Lymphocyte proliferation assays (LPA), which can provide important information regarding the immune reconstitution of human immunodeficiency virus (HIV)-infected patients on highly active antiretroviral therapy, frequently involve shipment of specimens to central laboratories. In this study, we examine the effect of stimulant, anticoagulant, cell separation, storage, and transportation on LPA results. LPA responses of whole blood and separated peripheral blood mononuclear cells (PBMC) to different stimulants (cytomegalovirus, varicella-zoster virus, candida and tetanus toxoid antigens, and phytohemagglutinin) were measured using fresh specimens shipped overnight and frozen specimens collected in heparin, acid citrate dextrose (ACD), and citrate cell preparation tubes (CPT) from 12 HIV-infected patients and uninfected controls. Odds ratios for positive LPA responses were significantly higher in separated PBMC than in whole blood from ACD- and heparin-anticoagulated samples obtained from HIV-infected patients and from ACD-anticoagulated samples from uninfected controls. On separated PBMC, positive responses were significantly more frequent in fresh samples compared with overnight transportation for all antigens and compared with cryopreservation for the candida and tetanus antigens. In addition, viral antigen LPA responses were better preserved in frozen PBMC compared with specimens shipped overnight. CPT tubes yielded significantly more positive LPA results for all antigens, irrespective of the HIV patient status compared with ACD, but only for the candida and tetanus antigens and only in HIV-negative controls compared with heparin. Although HIV-infected patients had a significantly lower number of positive antigen-driven LPA responses compared with uninfected controls, most of the specimen processing variables had similar effects on HIV-positive and -negative samples. We conclude that LPA should be performed on site, whenever feasible, by using separated PBMC from fresh blood samples collected in either heparin or ACD. However, if on-site testing is not available, optimal transportation conditions should be established for specific antigens.

Adult↗
PubMed2003

Elevated results in a vitamin B12 assay when using serum separator blood collection tubes.

BACKGROUND: In the light of apparently spurious serum vitamin B(12) results in some patients, the effect of serum separator sample tubes on serum vitamin B(12) values, assayed by the Bayer Centaur analyser, was examined. METHOD: Results of parallel assays of serum vitamin B(12) in plain (non-gel) serum tubes and serum separator gel tubes were compared. Serum in previously centrifuged gel tubes was mixed in the tube and the effect of that mixing on assay results quantitated. A limited investigation of the effect of tube cap type was also carried out. RESULTS: Serum vitamin B(12) concentration was 54% higher in samples taken into serum separator tubes after re-mixing the serum in the original tube. This effect could be abolished by re-centrifugation. CONCLUSIONS: Use of serum separator tubes may be associated with spuriously elevated serum vitamin B(12) concentrations in the Bayer Centaur assay. Laboratories receiving samples for vitamin B(12) assay in serum separator tubes that have already been centrifuged should either re-centrifuge the tubes, or aliquot and re-centrifuge serum from the tubes prior to vitamin B(12) assay.

Artifacts↗
PubMed2005

Early separation anxiety and adult attachment style in women with eating disorders.

OBJECTIVE: To ascertain whether women with eating disorders have a higher frequency of separation anxiety symptoms in childhood, and a higher prevalence of insecure styles of adult attachment, compared with healthy women. METHODS: The Separation Anxiety Symptom Inventory (SASI) and the Attachment Style Questionnaire (ASQ) were administered to 78 women with eating disorders and 64 healthy women. RESULTS: Compared with control women, women with eating disorders reported more severe symptoms of separation anxiety during childhood, and scored higher on the ASQ scales, reflecting insecure styles of adult attachment. Early separation anxiety and insecure attachment were not correlated with age of onset or illness duration. The diagnostic subgroup was not associated with a specific style of insecure attachment: compared with control women, both anorexic and bulimic women scored higher on the ASQ scales reflecting anxious attachment, but not on the scales reflecting avoidant attachment. CONCLUSIONS: The results confirm the link between eating disorders and insecure attachment that has been found in previous studies, and extend it to childhood symptoms of separation anxiety.

Adolescent↗
PubMed2000

Long-term biobehavioral effects of maternal separation in the rat: consistent or confusing?

Over the last decades of research there has been increasing interest in endocrine and behavioral effects of postnatal environmental manipulations. A manipulation procedure that has been widely used to date is that of maternal separation. Many studies have demonstrated that, in the rat, a single or repeated separation of the pups from the mother leads to acute as well as long-term effects on endocrinology and behavior. However, reviewing the literature shows that contrary findings for almost all parameters investigated can be found. A possible explanation for this inconsistency may be the fact that maternal separation has become a collective term for a variety of extremely different experimental manipulations. Therefore, this review aims at evaluating typical effects of maternal separation in the laboratory rat by categorizing different experimental procedures. We concentrate in particular on longterm behavioral effects, although a brief summary of neuroendocrine effects is also provided. In addition, important methodological issues of maternal separation studies are discussed as a possible source for inconsistent findings.

Adaptation, Physiological↗
PubMed2001

Frequency of nonspecific clinical signs in dogs with separation anxiety, thunderstorm phobia, and noise phobia, alone or in combination.

OBJECTIVE: To determine the frequency of nonspecific clinical signs in dogs with separation anxiety, thunderstorm phobia, noise phobia, or any combination of these conditions and determine whether these conditions are associated in dogs. DESIGN: Case series. ANIMALS: 141 dogs. PROCEDURE: Diagnoses were established using specific criteria. Owners of dogs completed a questionnaire on how frequently their dogs exhibited destructive behavior, urination, defecation, vocalization, and salivation when the owners were absent and the types and frequency of reactions to thunderstorms, fireworks, and other noises. RESULTS: Associations of the 3 conditions and of various nonspecific clinical signs within and between diagnoses were nonrandom. The probability that a dog would have separation anxiety given that it had noise phobia was high (0.88) and approximately the same as the probability it would have separation anxiety given that it had thunderstorm phobia (0.86). However, the probability that a dog would have noise phobia given that it had separation anxiety (0.63) was higher than the probability that it would have thunderstorm phobia given that it had separation anxiety (0.52). The probability that a dog would have noise phobia given that it had thunderstorm phobia (0.90) was not equivalent to the converse (0.76). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that dogs with any of these conditions should be screened for the others. Interactions among these conditions are important in the assessment and treatment of dogs with > 1 of these conditions. Responses to noise were different from those to thunderstorms, possibly because of the unpredictability and uncertainty of thunderstorms.

Animals↗
PubMed1993

Reported early separation anxiety symptoms in patients with panic and generalised anxiety disorders.

Attachment theory has proposed that early separation anxiety is a risk factor for adult anxiety disorder, with the recent focus being particularly on panic disorder. The results of empirical studies examining this link are, however, contradictory, possibly because of inconsistencies across studies in measuring memories of early separation anxiety. In the present study, a psychometrically sound measure, the Separation Anxiety Symptom Inventory (SASI) was used to compare memories of such early symptoms in panic disorder (including those with mild phobic-avoidance), generalised anxiety disorder and control subjects. Anxiety patients as a group returned higher SASI scores (p < 0.001) with a non-significant trend for panic disorder patients to score higher than those with generalised anxiety. These results suggest that early separation anxiety may be a harbinger of adult anxiety and that risk of panic disorder may be higher in the most severely affected youngsters. As a risk factor, early separation anxiety does not however appear to be uniquely related to adult panic disorder.

Adult↗
PubMed1994

Effects of different density gradient separation techniques on neutrophil function.

Investigation of the pathogenesis of inflammatory diseases has involved assessment of polymorphonuclear leukocyte (PMN) activity and a variety of techniques for separating PMNs from whole blood have been described. In this study the effects of Percoll gradient, Ficoll-Hypaque/Dextran sedimentation (FH/DS) and Mono-Poly Resolving Medium (M-PRM) on activation and function of PMNs were compared. All three separation techniques gave similar cell yield and purity. The mean (+/- SEM) percentage of cells demonstrating pseudopodia formation for Percoll, FH/DS and M-PRM were 39 +/- 9, 57 +/- 6 and 63 +/- 5, respectively, while superoxide release from resting cells was 1.9 +/- 0.9, 7.2 +/- 3.5 and 11 +/- 4.8 pmols per 10(6) cells min-1, respectively, indicating that activation of cells during separation may be less with Percoll compared to the other methods. The functional capacity of the cells to respond to a stimulus was similar for all methods as indicated by similar EC50 values for chemotaxis to zymosan-activated serum and similar superoxide production induced by tetradecanoyl phorbol acetate. All three separation techniques produce functionally active PMNs of high purity but the use of Percoll gradients may be preferable when a quick method of separation which causes minimum pre-activation of PMNs is required.

Cell Separation↗
PubMedDate not supplied

Real-time sperm separation system: a review of Wang tubes and related technologies.

This paper discusses real-time sperm separation technology and the use of Wang tubes for isolating high-quality and fertile sperm. Several methods have been developed and used to prepare a better sperm specimen, including the conventional swim-up technique, Percoll discontinuous density gradient, Sephadex gel filtration, centrifugation, migration-sedimentation, albumin column, and Sperm-prep. However, none of these methods has the ability to isolate from the original ejaculum a large number of highly motile, morphologically normal, and normal-chromatin sperm, while retaining in an adequate volume of physiological medium, free of less-motile and nonmotile sperm, and free of microorganisms, cell debris, seminal plasma, leukocytes, and foreign bodies. These methods should be classified as incomplete techniques of sperm separation and are of only limited effectiveness in infertility treatment. They may also cause irreparable damage to the mother or fetus. Real-time sperm separation technology is based on the observations that nonpathological spermatozoa do not transfer organisms, that the motility pattern and swim-up capacity of pathological sperm are limited or disturbed, and that the movement of sperm differs from the passive motion of nonciliated microorganisms and the random active motion of ciliated microorganisms in terms of velocity and direction. Real-time sperm separation technology encompasses the Wang tube system, which has two main functions: to isolate organisms-free, normal-chromatin, morphologically normal, and highly motile spermatozoa that are suitable for use in infertility treatment, and to prevent sex-linked inherited diseases. The results of these evaluations indicate that real-time sperm separation technology is the most effective and precise method of isolating and decontaminating sperm to date.

Cell Separation↗