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PubMed1990

Biochemical and physiological alterations in canine neutrophils separated by lysis or Percoll gradient isolation technologies.

Neutrophils were isolated from the peripheral blood of clinically normal canines by hypotonic lysis or density gradient (Percoll) centrifugation techniques. As a function of preparative technique, separated neutrophils were examined in vitro for alterations in membrane lipid integrity, and both granular and cytosolic specific enzymes. Membrane lipid disorder was assessed by merocyanine 540 (MC540), a fluorescent bioprobe, which intercalates into disrupted cellular lipid membranes. Evidence of membrane lipid disorder was based upon comparisons of mean cellular fluorescence exhibited by unstimulated cells. Based upon comparisons of mean cellular fluorescence the isolation methodologies did not appear significantly different. Significant levels of membrane disruption, due to preparative technique, were evident upon neutrophil stimulation with phorbol myristate acetate (PMA). Neutrophils which were Percoll-separated and PMA-stimulated demonstrated significant levels of membrane disruption not apparent in lysis-separated stimulated aliquots of cells. Cellular isolation methods did not significantly alter cellular myeloperoxidase (MPO) levels based on comparisons of cellular totals. Extracellular lactate dehydrogenase (LDH) levels of lysis-separated cells were four-times those of Percoll-separated neutrophils.

Animals↗
PubMed1999

[Collection of umbilical cord blood to separate the peripheral blood stem cells during the third stage of labor].

In order to collect the umbilical cord blood for the separation of peripheral blood stem cells, obstetrician have to get the document of informed consent from the pregnant women for the umbilical cord blood donation during pregnancy. After the fetal delivery, umbilical cord blood is easier collected from the umbilical vein of the placenta in utero than that of the delivered placenta. After the sterilization of umbilical cord, the special bag attached with tube and needle is more useful to collect the blood disinfectively and abundantly than the syringe. Blood stem cells should be separated as soon as possible after collection, but umbilical blood may be stored in the refrigerator until 24 hours after the collection, if the immediate separation is impossible. There is the positive correlation between the collected blood volume or the preserved hours until the separating management and the separated stem cells. Hearing on the health of the child should be followed until 6 months after delivery.

Cell Separation↗
PubMed1999

Bovine spermatozoal head size variation and evaluation of a separation technique based on this size.

The objectives of this study were to determine the variation of head areas of normal spermatozoa attributable to breed, individual bull and ejaculate and to verify separation of X and Y chromosome-bearing spermatozoa and separation effectiveness. Spermatozoa were evaluated using video enhanced contrast microscopy combined with video intensified fluorescent microscopy and the polymerase chain reaction (PCR). In Experiment 1, spermatozoal head areas were measured from 2 ejaculates collected from bulls of 3 beef and 2 dairy breeds. No differences in head areas were found between breeds or between bulls within breeds; variation was observed among ejaculates from individual bulls across breeds. In Experiment 2, spermatozoa from 5 ejaculates were separated on individual SEPDEVICEs (Patented). Head area, fluorescent intensity and PCR of spermatozoa retained in the SEPDEVICEs suggested a separation based on size in 1 of 5 samples. Ejaculate variation in head areas affected separation efficiency.

Animals↗
PubMed1975

PHA stimulation of separated human lymphocyte populations.

Lymphocyte preparations from peripheral blood and tonsils were separated into populations enriched with T or B cells by formation of rosettes with SRBC and separation of the rosette-forming and non-rosette-forming populations. T cell-enriched populations were also prepared by nylon column filtration. Using these methods preparations were obtained which comprised 80--95% T or B lymphocytes as determined by E-rosette formation and surface immunoglobulin (Ig) staining. PHA responsiveness, measured by [3H]thymidine incorporation, varied between relatively wide limits and was critically dependent on the degree of separation obtained. Relatively pure B-cell populations (less than 12% T cells) from blood and tonsils gave low PHA responses while preparations from blood still containing 24--38% T cells gave responses equal to or even greater than those of unseparated controls (60--78% T cells). T cell-enriched populations (80--86% T cells) responded to an equal or greater degree than controls but more efficient separation (greater than 90% T cells) resulted in markedly reduced stimulation. There was thus no simple correlation between the degree of phytomitogen-induced transformation and the number of T cells present. It is concluded that the low response of relatively pure T-cell populations may be due to depletion of B cells or non-lymphoid cells (or both) during the separation procedures. These observations have implications for the use of PHA stimulation as a measure of T-cell activity in mixed populations such as those of human peripheral blood leucocytes.

B-Lymphocytes↗
PubMed1975

Concurrent separation of plasma and of viable blood cells.

Fractions of viable, morphologically intact erythrocytes, leukocytes, and platelets were isolated concurrently with plasma from single samples of human blood. The four compartments were separated immediately after the collection of blood as well as at different times up to 120 minutes after the drawing. In addition, the cellular separation was applied at various intervals after the initial removal of plasma. All steps of the procedure were carried out at room temperature using simple laboratory equipment. The cross-contamination of the cellular fractions, obtained in yields ranging from 33 per cent to 69 percent was less than 5 per cent. More than 96 per cent of the isolated cells excluded trypan blue and retained lactate dehydrogenase. Regardless of the conditions of separation, the cellular contents of potassium in erythrocytes, leukocytes, and platelets were constant, averaging 270, 387, and 209 nEq. per milligram of protein, respectively. The isolated cells also maintained high ratios of cellular K+/Na+, except after their delayed isolation following the initial separation of plasma. Under the latter conditions, the cells were characterized by a gain in cellular sodium despite an unchanged content of potassium. The separation procedure was routinely applied to 5 to 10 ml. samples of blood, obtained from randomly selected volunteer subjects.

Blood Cell Count↗
PubMed2000

Comparative study on efficacy of three sperm-separation techniques.

AIM: To evaluate the comparative effectiveness of real-time sperm separation technique (Wang's tube method) and other two conventional methods in isolating high-quality sperm preparation, and to compare the spouse pregnancy rate in intrauterine insemination (IUI) with sperm preparations isolated by these methods. METHODS: The effectiveness of the real-time sperm separation technique, the conventional swim-up and the Percoll discontinuous density gradient methods in isolating sperm preparations from 60 infertile patients (20 with apparently normal semen and 40, abnormal semen contaminated with microorganisms and other impurities) was evaluated and compared. The microorganisms to be removed included bacteria, virus, Chlamydia trachomaticum, Ureaplsama urealyticum, etc. The spouse pregnancy rates in IUI with sperm preparations isolated by these three techniques from 80 oligoasthenoteratospermic patients were also compared. RESULTS: The quality (including the percentages of normal form, normal-chromatin and motile sperm, and the grade of motility) of sperm obtained by the real-time sperm separation technique was much higher (P < 0.01) as compared with those by the other two methods. The Wang's tube method was also more effective in removing microorganisms and other impurities. The method provided a higher IUI pregnancy rate than the other two sperm separation techniques (P < 0.05). CONCLUSION: The real-time sperm separation technique is the most effective method so far available in isolating high-quality sperm samples to be used in assisted reproduction.

Adult↗
PubMed2002

[The effect of separating red blood cells from bone marrow graft in vitro by methylcellulose].

It has need to separate red blood cells (RBC) from marrow graft in ABO group unmatched BMT and auto-BMT with purging tumor cells, the separating effect of methylcellulose was observed. The mixture of 0.5% methylcellulose and bone marrow was laid up in an open transfusion system, and then sedimentation of RBC was performed in the transfusion tube. The separating results of 18 marrow grafts showed that the recovery rates of mononuclear cells and CD34(+) cells were (83.8 +/- 55.2)% and (90.3 +/- 7.2)%, respectively. RBC residual rate was (4.3 +/- 1.5)%. The yield of CFU-GM was (60.8 +/- 22.4)/2 x 10(5) MNC, and there was no difference to [(69.8 +/- 23.4)/2 x 10(5) MNC] yielded from same marrow samples, separated by Ficoll-Hypaque separation. It is concluded that this method could be used for bone marrow transplantation.

Bone Marrow Transplantation↗
PubMed2005

Intramuscular ketamine pre-medication to separate children from their parents.

Total of 68 cases (children of age ranging from 1-10 years) were analyzed to see the effect of intramuscular ketamine to separate them from their parents. Every case was injected ketamine 5mg/kg of body weight and atropine 0.02mg/kg of body weight. The result showed 56 (82.4%) of the cases to be asleep, 10 (14.7%) of the cases calmly separated, 2 (2.9%) of the cases sedated but tearing and none of them was found difficult to separate from the parents. Tolerance of face mask and IV opening showed, 60 (88.24%) of the cases easily tolerated the face mask and IV opening, 6 (8.8%) of the cases slightly moved and 2 (2.9%) of the cases had to be held by assistant during application of face mask and IV opening. The pulse rate slightly increased after 5 minutes of pre-medication and slowed down after 10 minutes more or less to the baseline, and after 15 minutes pulse rate slightly dropped beyond the base line showing that patients were more haemodynamically stable. As pulse rate before pre-medication was found slightly higher than after 15 minutes of pre-medication it may due to apprehension and anxiety. Average time taken to separate the patients was 7.1 minutes (Range, 5 to 10 minutes) which is very convenient for conducting the operation in time. From this study it was concluded that Intramuscular Ketamine for pre-medication in children to separate from the parents is effective, takes less time, keeps the patients haemodynamically stable and is very safe.

Anxiety, Separation↗
PubMed1991

Separation of periportal and perivenous rat hepatocytes by fluorescence-activated cell sorting: confirmation with colloidal gold as an exogenous marker.

Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion, respectively, we separated the isolated hepatocytes on a fluorescence-activated cell sorter. The common way to check on proper separation is to estimate activities of enzymes known to exhibit a heterogeneous acinar distribution. Using enzyme histochemistry, however, we found that already on short collagenase perfusion, some enzymes displayed a more shallow gradient than in vivo, making enzyme activities less suitable as zonal markers. We therefore used colloidal gold granules (17 nm) injected intravenously (2.5 mg) into the rat 2 to 3 hr before cell isolation. The gold is taken up predominantly by perivenous hepatocytes, probably because of the efficient removal of gold granules in zone 1 by competing Kupffer cells. We compared acridine orange fluorescence, presence of gold particles and activities of six marker enzymes, three biochemically and three histochemically determined. Acridine orange and gold both pointed to a high enrichment of the fractions, whereas most enzyme activities were more randomly distributed among the cells as a result of the isolation procedure. Our separation procedure yielded fractions highly enriched in either viable periportal or perivenous cells, both from one liver. The use of colloidal gold as a marker to monitor separation is a valuable alternative to the more risky estimation of enzyme activities.

Acridine Orange↗
PubMed1985

Zinc content of cellular components of blood: methods for cell separation and analysis evaluated.

Platelets, mononucleated cells, polymorphonucleated cells, and erythrocytes were separated from whole blood by use of discontinuous gradients of colloidal polyvinylpyrrolidone-coated silica ("Percoll"). We measured the zinc content of these cells by flame atomic absorption spectrophotometry, using a modified technique for micro-samples that obviated matrix interferences. Thus, results obtained by conventional flame atomic absorption and by the micro-method were identical. Inter-comparisons of separation methods indicated that separation of platelets and mononucleated cells by a two-gradient system of "Ficoll-Hypaque" (a synthetic polymer of sucrose) or Percoll was relatively poor, whereas there was a good separation when a tertiary gradient system of Percoll was used. The apparent zinc content of mononucleated cells depended on the degree of separation from the platelets, with contamination by platelets resulting in artificially high values for mononucleated cells.

Adult↗
PubMed1979

Separation of normal human bone marrow cells by counterflow centrifugal elutriation. I. Morphological analysis and subfractionation of neutrophilic granulocytes.

We have used counterflow centrifugal elutriation to separate the nucleated cells of normal human bone marrow into their major morphologically identifiable cellular components. Human bone marrow cells were separated simultaneously into the major cell types--lymphocytes, monocytes, granulocytes, and erythroid precursors. Significant concentration of bone marrow cell types could be achieved, including myeloblasts (x 25), promyelocytes (x 43), early myelocytes ( x 22), late myelocytes (x 17), metamyelocytes (x 6), eosinophils (x 40), basophils (x 55), erythroblasts (x 35), monocytes (x 40), and plasma cells (x 35). Cell loss is minimal (85% recovery of input) and random, and cell viability, as measured by trypan blue staining, is high (greater than 95%). Factors interfering with separation are also described: a low loading flow rate, the presence of an excess of mature red cells, and the absence of fetal calf serum resulted in poor cell recovery and viability. The use of heparin as the anticoagulant resulted in cell loss, decrease in cell viability, and an ineffective separation. Separation of cells from normal bone marrow by centrifugal elutriation should facilitate the study of bone marrow cell interactions and the purification of individual types of bone marrow cells.

Bone Marrow↗
PubMed1978

Separation of rabbit marrow precursor cells by combined isopycnic sedimentation and electronic cell sorting.

Separation of developing cells into fractions of differing stages of maturity is critical to effective biochemical study of the process of cellular differentiation. Density gradient techniques utilizing rate-zonal or isopycnic separations have permitted partial separations based on cell mass or cell density. In this study the separation of various rabbit marrow cells was improved by high-speed flow analysis and sorting in a Coulter Two-Parameter Cell Sorter. After preliminary isopycnic separation of marrow cells in Ficoll-Hypaque, cells were sorted into lymphoid and myeloid elements, utilizing light-scatter (LS) profiles to determine sorting. Characteristic LS patterns were present for erythrocytes, lymphocytes, devitalized cells, and granulocytes. When erythrocytes and their precursors were removed by hypotonic lysis, the remaining granulocytes could be sorted to give samples with much greater purity with respect to developmental stage than was possible with density gradients alone. Thus the combination of techniques represents a significant improvement in the ability to study the mechanisms of normal or altered cellular maturation.

Animals↗
PubMed1982

Initial evaluation of a convection counter streaming galvanization technique of sex separation of human spermatozoa.

A new method for separating X and Y human spermatozoa called convection counter streaming galvanization was evaluated. The method was independently performed by this semenology laboratory with the use of the special separation equipment and extending media provided by its developer, Dr. Bhairab C. Bhattacharya. The mean number of Y spermatozoa increased from 48% to 77% in the separated fraction predicted to be Y-enriched. The fraction predicted to be X-enriched increased from a mean of 52% to 77%. The one separation process allowed accumulation of both enriched fractions simultaneously. The separated portions of spermatozoa maintained good motility and penetration of cervical mucus but produced a mean recovery concentration in the X- and Y-enriched fractions of only 15% to 16% of the preseparation concentration.

Cell Separation↗
PubMed1994

An immunomagnetic separation method using superparamagnetic (MACS) beads for large-scale purification of human mammary luminal and myoepithelial cells.

A comparison has been made between different immunomagnetic techniques for separating normal human mammary epithelial cells based on the exclusive expression of EMA (epithelial membrane antigen) by luminal cells and CALLA (CD10) by myoepithelial cells. When cells labelled with antibodies to these antigens were incubated with Dynabeads, they rosetted myoepithelial but not luminal cells. However, both luminal and myoepithelial cells could be positively separated using the MACS system. Purity was established by analyzing the expression of CALLA and EMA using flow cytometry, and of cell-type specific cytokeratins using indirect immunofluorescence. Dynabead-separated myoepithelial cell populations were of high purity (> 98%) but the beads could not be removed from the cells. Luminal cell populations separated by the MACS method were also highly purified (> 95%), as were myoepithelial cell populations (> 90%). Using this immunomagnetic separation method, up to 10(7) cells of each type could be obtained from individual preparations.

Adolescent↗
PubMed1993

[Preliminary results of the 2nd multicenter study. Thrombocytapheresis with the Fresenius AS-104 blood cell separator and ring study of cell counting. Section of Therapeutic and Preparative Hemapheresis of the DGTI].

Only multicenter studies on cell separators give valid data to compare different cell separators. The aim of the 2nd multicenter study was to evaluate the separation protocol, software version V 4.61, of the Fresenius AS-104 cell separator for efficiency and deviation from predicted yields. Plateletpheresis data from twelve hemapheresis centers, using identical apheresis protocols and cell counting methods, were registered and statistically analyzed. Additionally, the counting methods of the centers were controlled by a ring study with biweekly external cell count trials. To get a comparison, the apheresis data, which depend on the center effects, were corrected by the values from the ring study. Preliminary results of 380 runs are 45.2 +/- 8.1% for the separation effectivity, 1.95% deviation from the predicted yield, whereby 90% of all runs deviated less than 20% from the predicted yield. 50% of products had a WBC contamination below 6 x 10(6).

Blood Volume↗
PubMed1993

Lymphocyte phenotypes in infants are altered by separation of blood on density gradients.

Flow cytometry techniques for immunophenotyping have revolutionised the diagnosis and monitoring of paediatric immunological disorders. Although recent studies in adult subjects discourage the use of density gradients for cell preparation prior to phenotyping, these procedures continue to be used. The purpose of this study was to determine the effect of density gradient separation on lymphocyte phenotypes from neonates, infants, and adults as compared to whole blood determinations. Subset distributions were different with the two procedures. In all three groups, CD19+ (B cell) and CD8+ (suppressor/cytotoxic T cell) percentages were significantly lower and CD3-CD56+ (NK cell) percentages were significantly higher in the density gradient separated cells. The loss of CD8+ cells in density gradient separation was shown to be a selective event. The CD8+CD11b- (cytotoxic T) subset percentages were lower in the density gradient separated cells, while the percentages of CD8+CD11b+ (suppressor T) cells were not affected by separation procedure. Because of the selective loss of lymphocytes on density gradients, the use of a whole blood technique for immunophenotyping in paediatric subjects is recommended.

Adult↗
PubMed1995

[Comparison of platelet and leukocyte content in platelet concentrates obtained from CS-3000 and third generation (CS-3000 plus and Cobe-Spectra) cell separators].

Platelet concentrates (PCs) obtained using old generation of cell separators contain high number of leukocytes. White cell (WBC) contamination and platelet (Plts) number in PCs obtained from separator II-nd generation CS-3000 and separators III-rd generations CS-3000 plus and Cobe-Spectra have been determined. PCs from new separators contain the same Plts number as PCs from CS-3000. The average leukocyte count in PCs obtained from CS-3000 was 171.26 x 10(6), whereas WBC number in PCs from CS-3000 plus and Cobe-Spectra were 2.87 and 2.54 x 10(6) respectively. In about 85% of PCs obtained from III-rd generation cell separators the leukocyte count did not exceed 5 x 10(6). This count is considered sufficient to prevent alloimmunization of HLA antigens. The determination of WBC count in every PCs allows to select PCs with fewer than 5 x 10(6) leukocytes and to transfuse them without the necessity of using expensive filters for leukocyte removing.

Blood Platelets↗
PubMed1997

[Technical aspects of extraction of platelet concentrates from dogs with an automated cell separator].

The results of this work point out that the cell separator AS 104 (Fresenius AG, Bad Homburg), which is developed to obtain platelet concentrates from human beings, can be used for dogs. The base adjustment of the cell separator for human beings (adjustment I) is useful, because of the high yield [platelet count (median): 1.75 x 10(11)] and quality [low leucocyte- (40/microliter) and erythro count (10 x 10(3)/microliter)]. In addition, there was no clear difference between the base adjustment (adjustment I) and an empirical modification (adjustment II). The platelet count of the donor dog decreased during the separation by approximately 140,000/microliter and reached the initial value after four days. Supplementary, the time course of the haematocrit and the concentrations of albumin, total protein and total calcium of the donor dog were measured. It should be mentioned that the concentrations of ionized calcium distinctly decreased during the separation. Synchronously, the concentrations of ionized calcium and of citrat reached the starting-counts 3-4 hours after the ending of the separation.

Animals↗