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PubMedDate not supplied

Preparative continuous separation of biological particles by means of free-flow magnetophoresis in a free-flow electrophoresis chamber.

For sorting, cells or cellular components can specifically be labeled by antibody-coated magnetic beads. We have developed a device for continuous magnetic sorting based on the flow-chamber of a free-flow electrophoresis system. Magnetically labeled particles are injected into a given continuously flowing chamber buffer and pass an inhomogeneous magnetic field, configurated perpendicular to the flow direction. According to its magnetic moment, the magnetic material is deviated into the direction of the magnetic forces, while nonmagnetic material passes the field without interaction. The magnetic forces can be changed with the electrical current of the solenoids producing the magnetic field. As in the free-flow electrophoresis system, the particle fractions are collected in different vials. On-line control of the experiments can be performed by an optical scanning system. Experiments with model particles achieved a sorting purity of more than 99% at a rate of up to 5 X 10(8) particles per hour. In experiments with blood cells, a high enrichment of either B-or-T-lymphocytes was obtained. In contrast to free-flow electrophoresis, there is no limitation, in principle, regarding the type of chamber buffer to be used. This allows an optimal adaptation of the buffer conditions to the requirements of vital sorting. The preliminary results so far confirm this conclusion.

B-Lymphocytes↗

PubMedDate not supplied

Computational study of the effect of geometric and flow parameters on the steady flow field at the rabbit aorto-celiac bifurcation.

Arterial hemodynamic forces may play a role in the localization of early atherosclerotic lesions. We have been developing numerical techniques based on overset or "Chimera" type formulations to solve the Navier-Stokes equations in complex geometries simulating arterial bifurcations. This paper presents three-dimensional steady flow computations in a model of the rabbit aorto-celiac bifurcation. The computational methods were validated by comparing the numerical results to previously-obtained flow visualization data. Once validated, the numerical algorithms were used to investigate the sensitivity of the computed flow field and resulting wall shear stress distribution to various geometric and hemodynamic parameters. The results demonstrated that a decrease in the extent of aortic taper downstream of the celiac artery induced looping fluid motion along the lateral walls of the aorta and shifted the peak wall shear stress from downstream of the celiac artery to upstream. Increasing the flow Reynolds number led to a sharp increase in spatial gradients of wall shear stress. The flow field was highly sensitive to the flow division ratio, i.e., the fraction of total flow rate that enters the celiac artery, with larger values of this ratio leading to the occurrence of flow separation along the dorsal wall of the aorta. Finally, skewness of the inlet velocity profile had a profound impact on the wall shear stress distribution near the celiac artery. While not physiological due to the assumption of steady flow, these results provide valuable insight into the fluid physics at geometries simulating arterial bifurcations.

Algorithms↗

PubMed1992

High gradient magnetic separation of cells on the basis of expression levels of cell surface antigens.

The possibility of separating cells on the basis of levels of antigen expression was explored in a model system using fixed erythrocytes and high gradient magnetic separation (HGMS). Fixed human erythrocytes were labelled to varying degrees with tetrameric monoclonal antibody complexes specific for both dextran and glycophorin A-M. The cells were then mixed and incubated with dextran iron particles prior to magnetic separation. The small size of the dextran iron particles (less than 0.2 microns) resulted in quantitative magnetic labelling of cells as shown using fluoresceinated anti-dextran antibodies and flow cytometry. The relationships between the initial percentage of labelled cells, cell recovery, non-specific entrapment of unlabelled cells, the purity of the removed fraction, the degree of antigen expression and separation conditions (flow rate and field strength) were determined and used to establish separation conditions that allowed recovery of cells that differ only in the degree of antibody labelling.

Antibodies, Monoclonal↗

PubMed1995

Proliferation markers in breast carcinoma. Mitotic figure count, S-phase fraction, proliferating cell nuclear antigen, Ki-67 and MIB-1.

Proliferative rate is an important prognostic marker in breast carcinoma. However, the best measurement method has not been established. This study evaluated mitotic figure counts (MFC) as mitoses per 10 high power fields (HPF) and per 1,000 cells, S-phase fraction by flow cytometry, and Ki-67, MIB-1, and proliferating cell nuclear antigen (PCNA) positivity by immunohistochemistry in 135 breast carcinomas. There was strong correlation between the two MFC methods and significant correlation between MIB-1 positivity and all proliferation markers except Ki-67. S-phase fraction showed significant correlation with all proliferation markers except PCNA. Ki-67 positivity correlated only with S-phase fraction, and PCNA positivity only with MIB-1 and mitoses per 10 HPF. High MFC was associated with other prognostic factors: high histologic tumor grade, absence of biochemical and immunohistochemical hormone receptors, and DNA aneuploidy, but not lymph node involvement or tumor size. MIB-1 positivity was also associated with these parameters, except lymph node involvement, tumor size, and DNA aneuploidy. Mitotic figure count and MIB-1 positivity were associated strongly with disease-free survival, up to 46 months. The other proliferation markers were associated with fewer prognostic factors and showed weak or absent association with disease-free survival. The best proliferation markers are mitotic figure counting (either method) or MIB-1 positivity.

Antibodies, Monoclonal↗

PubMed2005

Field-scale evaluation of the passive flux meter for simultaneous measurement of groundwater and contaminant fluxes.

A new method, passive flux meter (PFM), has been developed and field-tested for simultaneously measuring contaminant and groundwater fluxes in the saturated zone at hazardous waste sites. The PFM approach uses a sorptive permeable medium placed in either a borehole or monitoring well to intercept contaminated groundwater and release "resident" tracers. The sorbent pack is placed in a groundwater flow field for a specified exposure time and then recovered for extraction and analysis. By quantifying the mass fraction of resident tracers lost and the mass of contaminant sorbed, groundwater and contaminant fluxes are calculated. Here, we assessed the performance of PFMs at the Canadian Forces Base Borden field site in Ontario, Canada. Two field tests were conducted under imposed groundwater flow fields: (1) radial flow to a well and (2) linear flow in a test channel confined by sheet pile walls on three sides. Both tests demonstrate that the local fluxes measured by PFM and averaged overthe screen interval were within 15% of imposed groundwaterflow and within 30% of measured contaminant mass flux. Patterns in depth variations in groundwater and contaminant fluxes, determined by the PFM approach, allow for site characterization at a higher spatial resolution. These results support the PMF method as a potential innovative alternative for measuring groundwater and contaminant fluxes in screened wells.

Alcohols↗

PubMed1994

[A description of a new method of quantifying the regurgitant flow in patients with an incompetent mitral valve].

BACKGROUND: It has been shown previously that using color Doppler flow mapping, simple measurements of apparent jet size do not correlate closely with regurgitant flow rate and regurgitant fraction. Recently the proximal flow convergence method has been proposed to quantify valvular regurgitation by analysis of the converging flow field proximal to a regurgitant lesion. Flow rate Q can be calculated as Q = 2 pi r2va where va is the aliasing velocity at a distance r from the orifice. PATIENTS AND METHODS: In 54 patients (43 in sinus rhythm, 11 with atrial fibrillation) with at least mild mitral regurgitation by semi-quantitative assessment, regurgitant stroke volume, regurgitant flow rate, and regurgitant fraction were calculated using the proximal flow convergence method and compared with values obtained by the Doppler/two-dimensional echocardiographic method. RESULTS: Regurgitant stroke volumes (VL) calculated by the proximal flow convergence method correlated very closely with values obtained by the Doppler-2D method with r = 0.93 (y = 0.95x + 0.55) and delta VL = -0.3 +/- 4.0 cm3. Regurgitant flow rates (Q) calculated by both methods showed a similar correlation: r = 0.93 (y = 0.95x + 54) and delta Q = -34 +/- 284 cm3/min. The correlation for regurgitant fraction (RF) calculated by both techniques showed r = 0.89 (y = 0.98x + 0.006) and delta RF = -0.005 +/- 0.06. All correlations were slightly better for the group of patients in sinus rhythm compared with the study group in atrial fibrillation. CONCLUSION: This study demonstrates that the proximal flow convergence method is an accurate and reproducible technique to quantify mitral regurgitation. This approach is easy and less time-consuming than the Doppler-echocardiographic method. While future improvements of this method are to be expected, flow calculations based on the assumption of simple hemispheric symmetry of the proximal flow field appear suitable for clinical application at the present time.

Adult↗

PubMed2006

Mobilization of river transported colloidal aluminium upon mixing with seawater and subsequent deposition in fish gills.

During flooding events, increased concentrations of gill-reactive aluminium (Al) have been identified in estuarine water causing high Al accumulation in fish gills. By in situ filtering and ultrafiltering river water (pH 5.5-6.4, 3-5 mg L(-1) DOC) and continually mixing the size fractioned river water with seawater (30 in salinity), Al mobilization was studied in flow-through tank systems (6 in salinity) during a six-week field experiment in Western Norway. Atlantic salmon (Salmo salar L.) kept in tanks were continuously exposed to the mixtures. Based on in situ Al fractionation of the experimental waters and sampling of gills from exposed fish, results showed that Al associated with river transported colloids, probably organic material ( 8 kDa) was mobilized to low molecular mass cationic Al-species (LMM Al(i)) upon contact with seawater. Mobilized Al(i)-species deposited immediately on fish gills. During high flow conditions with increased amounts of colloidal material, the concentration of mobilized LMM Al(i) and the Al accumulation in gills of fish exposed to river water-seawater mixtures increased by a factor of 5 and 10, respectively, compared to the input river water. The concentration of mobilized LMM Al(i) and the Al accumulation in fish gills decreased with time after mixing (from 1 to 30 min) and as high runoff subsided.

Aluminum↗

PubMed1995

Capillary perfusion pattern and microvascular geometry in heterogeneous hypoxic areas of hypoperfused rat myocardium.

The origin of heterogeneities in tissue oxygenation due to low-flow ischemia was studied in hypoperfused myocardium of anesthetized rats. In frozen sections of myocardial biopsies the localization of increases in NADH fluorescence, an indicator of tissue hypoxia, was compared with microvascular flow distribution and capillary geometry. The latter parameters were accomplished through capillary labeling with indicator dyes in vivo and enzyme-histochemical staining in vitro, respectively. Most NADH-fluorescent areas were found to have developed despite sustained capillary flow. When the fractions of arterial, venous, and intermediate capillary segments were analyzed within circumscribed hypoxic fields (< 200 microns diam), frequencies of 30.7 +/- 6.1, 35.3 +/- 5.3, and 30.8 +/- 5.0%, respectively, were found. In contrast, a significantly higher fraction of arterial segments (63.2 +/- 3.3%) and a lower percentage of venous segments (16.4 +/- 2.5%) were determined in nonhypoxic islands enclosed by hypoxic tissue. These results support the view that the latter zones are located near the arterial portion of the capillary bed where their oxygenation is favored during low-flow states. This effect appears to contribute to the supply heterogeneities in hypoperfused myocardium.

Animals↗

PubMed1995

Continuous focusing of biological particles by continuous immuno magnetic sorter: technique and applications.

We have developed a device for continuous deviation-mode, open-gradient fractionation of strongly magnetizable particles based on an electrophoresis counter-flow chamber. A mixture of magnetically labeled and nonlabeled particles can be injected into a given continuously flowing chamber buffer. The particles pass the inhomogeneous magnetic field of the open-gradient electromagnet in two narrow streams. According to the magnetic moments, induced by the magnetic field, magnetically labeled particles are deviated. The nonlabeled particles pass the magnetic field with negligible interaction. The deviated particles are focused into a stream that is completely separated from the streams of the nondeviated particles. The streams are fractionated by the counter-flow technique and collected in different vials. A high sorting purity, depending only on the specificity of the antibodies or similar labeling techniques, and a high through-put rate of up to 10(9) particles per hour was achieved. This was experimentally shown both by test particles and blood cells. The vital conditions of these blood cells were maintained by magnetic sorting.

B-Lymphocytes↗

PubMed2002

Numerical simulation and measurement of liquid hold-up in biporous media containing discrete stagnant zones.

We have studied hydrodynamic dispersion in single-phase incompressible liquid flow through a fixed bed made of spherical, permeable (porous) particles. The observed behaviour was contrasted with the corresponding fluid dynamics in a random packing of impermeable (non-porous) spheres with an interparticle void fraction of 0.37. Experimental data were obtained in the laminar flow regime by pulsed field gradient nuclear magnetic resonance and were complemented by numerical simulations employing a hierarchical transport model with a discrete (lattice Boltzmann) interparticle flow field. Finite-size effects in the simulation associated with the spatial discretization of support particles or dimension and boundaries of the bed were minimized and the simulation results are in reasonable agreement with experiment.

Biological Transport↗

PubMed2001

Free-flow zone electrophoresis: a novel approach and scale-up for preparative protein separation.

Different continuously working free-flow zone electrophoresis (FFZE) chambers have already been developed [1, 2]. All of them deal with the problem of distinctive Joule heating. The resulting temperature gradients cause an unstable density field which leads to thermal convection and thus to an intermixing of the different fractions within the chamber. The most promising and simple approach to stabilize the flow is to build chambers with one very small dimension (e.g., h = 0.5 mm) to assure efficient heat withdrawal. This in turn presents substantial disadvantages, namely limited throughput and restricted scale-up potential. The novel approach combines a simplified design and assembly with the possibility of straightforward scale-up. It still operates with one small dimension (d = 1-2 mm) to handle the Joule heating. Here, however, not the dimension perpendicular to the electric field but the dimension parallel to the electric field (separation distance) is chosen as the smallest dimension. The efficiency of the new device is shown by the separation of bovine serum albumin (BSA) and cytochrome c with an overall protein throughput of up to 1.1 g/h, using a cell with a separation volume of less than 20 mL.

Azo Compounds↗

PubMed1992

Quantification of mitral regurgitation with the proximal flow convergence method: a clinical study.

Accurate quantitation of valvular incompetence remains an important goal in clinical cardiology. It has been shown previously that when color flow Doppler mapping is used, simple measurements of apparent jet size do not correlate closely with regurgitant flow rate and regurgitant fraction. Recently the proximal flow convergence method has been proposed to quantify valvular regurgitation by analysis of the converging flow field proximal to a regurgitant lesion. Flow rate Q can be calculated as Q = 2 pi r2v(a), where v(a) is the aliasing velocity at a distance r from the orifice. In 54 patients (43 with sinus rhythm and 11 with atrial fibrillation) who had at least mild mitral regurgitation according to semiquantitative assessment, regurgitant stroke volume, regurgitant flow rate, and regurgitant fraction were calculated with the proximal flow convergence method and compared with values that were obtained by the Doppler two-dimensional echocardiographic method. Regurgitant stroke volumes (Vr) as calculated by the proximal flow convergence method correlated very closely with values that were obtained by the Doppler two-dimensional method, with r = 0.93 (y = 0.95x + 0.55) and delta Vr = -0.3 +/- 4.0 cm3. Regurgitant flow rates (Q) as calculated by both methods showed a similar correlation: r = 0.93 (y = 0.95x + 54) and delta Q = -34 +/- 284 cm3/min. The correlation for regurgitant fraction (RF) as calculated by both techniques showed r = 0.89 (y = 0.98x + 0.006) and delta RF = -0.005 +/- 0.06. All correlations were slightly better for the group of patients with sinus rhythm than for the study group of patients with atrial fibrillation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1995

31P NMR spectroscopy studies of phospholipid metabolism in human melanoma xenograft lines differing in rate of tumour cell proliferation.

The concentration of phospholipid metabolites in tumours has been hypothesized to be related to rate of cell membrane turnover and may reflect rate of cell proliferation. The purpose of the study reported here was to investigate whether 31P NMR resonance ratios involving the phosphomonoester (PME) or phosphodiester (PDE) resonance are correlated to fraction of cells in S-phase or volume-doubling time in experimental tumours. Four human melanoma xenograft lines (BEX-t, HUX-t, SAX-t, WIX-t) were included in the study. The tumours were grown subcutaneously in male BALB/c-nu/nu mice. 31P NMR spectroscopy was performed at a magnetic field strength of 4.7 T. Fraction of cells in S-phase was measured by flow cytometry. Tumour volume-doubling time was determined by Gompertzian analysis of volumetric growth data. BEX-t and SAX-t tumours differed in fraction of cells in S-phase and volume-doubling time, but showed similar 31P NMR resonance ratios. BEX-t and WIX-t tumours showed significantly different 31P NMR resonance ratios but similar fractions of cells in S-phase. The 31P NMR resonance ratios were significantly different for small and large HUX-t tumours even though fraction of cells in S-phase and volume-doubling time did not differ with tumour volume. None of the 31P NMR resonance ratios showed significant increase with increasing fraction of cells in S-phase or significant decrease with increasing tumour volume-doubling time across the four xenograft lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Shotgun polymerase chain reaction: construction of clone libraries specific to a NotI fragment of flow-sorted human chromosome 22.

We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.

Base Sequence↗

PubMed2000

Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.).

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.

Cell Cycle↗

PubMed2003

The heat shock-induced cell cycle arrest is attenuated by weak electromagnetic fields.

Stress-induced effects in human acute leukaemia cells (HL-60) were studied by flow cytometry using the fluorescent dye carboxyfluorescein succinimidyl ester which allows the analysis of several successive cell generations for up to 10 days. Asynchronously cycling cells subjected to heat shock (30 min at 41 degrees C) responded in two distinct ways: while one fraction of the cell population (about 15%) re-entered the cell cycle after a short delay, other cells became arrested at different phases of the cell cycle and remained arrested for up to several days and finally underwent apoptosis. Weak electromagnetic fields (60 micro T, 50 Hz) alleviated the heat-induced block and the fraction of arrested cells was significantly smaller.

Cell Cycle↗

PubMed1990

Purification of S1 nuclease from Aspergillus oryzae by recycling isoelectric focusing.

We describe the purification of a single-strand nuclease from Aspergillus oryzae using the first commercial prototype of an instrument (RF3TM) designed by Milan Bier et al. for preparative-scale isoelectric focusing. Protein separation takes place entirely in solution, with shear-stabilized laminar flow counteracting convective disturbances generated by the electric field. Conditions for isoelectric focusing were determined by focusing fractions with nuclease activity, following chromatography on DEAE-Sepharose, in analytical gels containing carrier ampholytes. The separation was then scaled up to process larger quantities of protein in the RF3. When partially-purified protein (250 mg, 6700 U/mg) was focused in pH 3-4 carrier ampholytes. 67% of the activity was recovered in pooled peak fractions with a specific activity of 54,000 U/mg protein. Overall, 82% of the activity loaded on the RF3 was recovered. Eliminating two steps prior to isoelectric focusing, and increasing the protein load from 250 mg to 1.2 g, produced an enzyme with a nearly four-fold increase in specific activity (from 4000 U/mg protein to 15,000 U/mg protein) but with unacceptable color. Our results indicate that a high quality enzyme can be prepared in quantity when heat denaturation and ammonium sulfate precipitation are included prior to isoelectric focusing.

Ammonium Sulfate↗

PubMed1990

Carcinoma of the breast in children.

Carcinoma of the breast in children is an extremely rare disease, only 44 patients have been reported in the world literature. We report on a case of breast cancer in a 9-year-old girl, who was admitted to the hospital because of a four-month history of a painless mass in the right breast. Clinical examination and mammography resulted in a strong suspicion of malignancy. The histology corresponded to juvenile secretory carcinoma, the histological type without the characteristics of prepubertal cancer cases. Electron microscopy demonstrated intracellular large vacuoles and acini filled with secretory material. The mitotic index was low, the highest measured value was 4 mitotic figures per 10 high power fields corresponding to 2.78 mitotic figures per mm2 of epithelium in the microscope field. Flow cytometry showed that the neoplastic cells were diploid and had a low S-phase fraction. A simple mastectomy including axillary evacuation was performed. No lymph node or distant metastases were found. Postoperative radiation treatment or chemotherapy were not administered. Our patient has been in follow-up for six years without recurrence. In this case simple mastectomy with axillary evacuation seems to have given a cancer-free development into adulthood. Earlier reports have also shown a favourable outcome in these tumours.

Breast↗