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Cleveland, OH, United States

Moore L.R.,Cleveland Clinic | Williams P.S.,Cambrian Technologies Inc. | Nehl F.,TU Dresden | Abe K.,Mie University | And 2 more authors.
Analytical and Bioanalytical Chemistry | Year: 2014

Emerging applications of rare cell separation and analysis, such as separation of mature red blood cells from hematopoietic cell cultures, require efficient methods of red blood cell (RBC) debulking. We have tested the feasibility of magnetic RBC separation as an alternative to centrifugal separation using an approach based on the mechanism of magnetic field-flow fractionation (MgFFF). A specially designed permanent magnet assembly generated a quadrupole field having a maximum field of 1.68 T at the magnet pole tips, zero field at the aperture axis, and a nearly constant radial field gradient of 1.75 T/mm (with a negligible angular component) inside a cylindrical aperture of 1.9 mm (diameter) and 76 mm (length). The cell samples included high-spin hemoglobin RBCs obtained by chemical conversion of hemoglobin to methemoglobin (met RBC) or by exposure to anoxic conditions (deoxy RBC), low-spin hemoglobin obtained by exposure of RBC suspension to ambient air (oxy RBC), and mixtures of deoxy RBC and cells from a KG-1a white blood cell (WBC) line. The observation that met RBCs did not elute from the channel at the lower flow rate of 0.05 mL/min applied for 15 min but quickly eluted at the subsequent higher flow rate of 2.0 mL/min was in agreement with FFF theory. The well-defined experimental conditions (precise field and flow characteristics) and a well-established FFF theory verified by studies with model cell systems provided us with a strong basis for making predictions about potential practical applications of the magnetic RBC separation. [Figure not available: see fulltext.] © 2013 Springer-Verlag Berlin Heidelberg.

Williams P.S.,Cambrian Technologies Inc.
Analytical and Bioanalytical Chemistry | Year: 2015

In asymmetrical flow field-flow fractionation (As-FlFFF), only the membrane-covered accumulation wall is permeable to fluid; the opposite channel wall is impermeable. Fluid enters the channel at the inlet and exits partly through the membrane-covered accumulation wall and partly through the channel outlet. This means that not only does the volumetric channel flow rate decrease along the channel length as fluid exits through the membrane but also the cross-channel component to fluid velocity must approach zero at the impermeable wall. This dependence of cross-channel fluid velocity on distance across the channel thickness influences the equilibrium concentration profile for the sample components introduced to the channel. The concentration profile departs from the exponential profile predicted for the ideal model of field-flow fractionation. This influences both the retention ratio and the principal contribution to bandspreading - the nonequilibrium contribution. The derivation of an equation for the nonequilibrium bandspreading parameter χ in As-FlFFF is presented, and its numerical solution graphed. At high retention, it is shown that the solutions for both retention ratio R and χ converge on those for the ideal model, as expected. At lower levels of retention, the departures from the ideal model are significant, particularly for bandspreading. For example, at a level of retention corresponding to a retention parameter λ of 0.05, R is almost 4 % higher than for the ideal model (0.28047 as compared to 0.27000) but the value of χ is almost 60 % higher. The equations presented for both R and χ include a first-order correction for the finite size of the particles - the steric exclusion correction. These corrections are shown to be significant for particle sizes eluting well before steric inversion. For example, particles of half the inversion diameter are predicted to elute 25 % slower and to show almost 40 % higher bandspreading when steric effects are not accounted for. The work presented contributes to the fundamental theory of As-FlFFF and allows quantitative prediction of both retention and bandspreading at all levels of retention. © 2015 Springer-Verlag.

Joshi P.,Cleveland Clinic | Williams P.S.,Cleveland Clinic | Williams P.S.,Cambrian Technologies Inc. | Moore L.R.,Cleveland Clinic | And 4 more authors.
Analytical Chemistry | Year: 2015

Connective tissue progenitors (CTPs) are a promising therapeutic agent for bone repair. Hyaluronan, a high molecular mass glycosaminoglycan, has been shown by us to be a suitable biomarker for magnetic separation of CTPs from bone marrow aspirates in a canine model. For the therapy to be applicable in humans, the magnetic separation process requires scale-up without compromising the viability of the cells. The scaled-up device presented here utilizes a circular Halbach array of diametrically magnetized, cylindrical permanent magnets. This allows precise control of the magnetic field gradient driving the separation, with theoretical analysis favoring a hexapole field. The separation vessel has the external diameter of a 50 mL conical centrifuge tube and has an internal rod that excludes cells from around the central axis. The magnet and separation vessel (collectively dubbed the hexapole magnet separator or HMS) was tested on four human and four canine bone marrow aspirates. Each CTP-enriched cell product was tested using cell culture bioassays as surrogates for in vivo engraftment quality. The magnetically enriched cell fractions showed statistically significant, superior performance compared to the unenriched and depleted cell fractions for all parameters tested, including CTP prevalence (CTPs per 106 nucleated cells), proliferation by colony forming unit (CFU) counts, and differentiation by staining for the presence of osteogenic and chondrogenic cells. The simplicity and speed of the HMS operation could allow both CTP isolation and engraftment during a single surgical procedure, minimizing trauma to patients and lowering cost to health care providers. © 2015 American Chemical Society.

Vastamaki P.,University of Helsinki | Vastamaki P.,VTT Technical Research Center of Finland | Williams P.S.,Cambrian Technologies Inc. | Jussila M.,University of Helsinki | And 2 more authors.
Analyst | Year: 2013

A theoretical and experimental study of continuous two-dimensional thermal field-flow fractionation (2D-ThFFF) is presented. Separation takes place in radial flow between two closely spaced discs, one of which is heated and the other cooled in order to maintain a temperature gradient across the channel. The cooled disc, which serves as the accumulation wall, is rotated relative to the other to create a shear component to the fluid flow. Under the influence of the thermal gradient and flow components, the sample components spiral outwards along different paths to the outer rim of the channel to be collected. The general principle of operation is described and an approximate theoretical model formulated for predicting the outlet position for the path of each sample component. The influence of the principal operational parameters, such as radial and angular flow rates and thermal gradient, on the deflection angle of the sample trajectory is investigated. Fractionation is demonstrated for polystyrene polymer standards in a binary solvent consisting of cyclohexane and ethylbenzene. Experimental results are compared with theoretical predictions. This journal is © The Royal Society of Chemistry 2014.

Williams P.S.,Cambrian Technologies Inc.
Analytical and Bioanalytical Chemistry | Year: 2016

Asymmetrical flow field-flow fractionation (As-FlFFF) has become the most commonly used of the field-flow fractionation techniques. However, because of the interdependence of the channel flow and the cross flow through the accumulation wall, it is the most difficult of the techniques to optimize, particularly for programmed cross flow operation. For the analysis of polydisperse samples, the optimization should ideally be guided by the predicted fractionating power. Many experimentalists, however, neglect fractionating power and rely on light scattering detection simply to confirm apparent selectivity across the breadth of the eluted peak. The size information returned by the light scattering software is assumed to dispense with any reliance on theory to predict retention, and any departure of theoretical predictions from experimental observations is therefore considered of no importance. Separation depends on efficiency as well as selectivity, however, and efficiency can be a strong function of retention. The fractionation of a polydisperse sample by field-flow fractionation never provides a perfectly separated series of monodisperse fractions at the channel outlet. The outlet stream has some residual polydispersity, and it will be shown in this manuscript that the residual polydispersity is inversely related to the fractionating power. Due to the strong dependence of light scattering intensity and its angular distribution on the size of the scattering species, the outlet polydispersity must be minimized if reliable size data are to be obtained from the light scattering detector signal. It is shown that light scattering detection should be used with careful control of fractionating power to obtain optimized analysis of polydisperse samples. Part I is concerned with isocratic operation of As-FlFFF, and part II with programmed operation. [Figure not available: see fulltext.] © 2016 Springer-Verlag Berlin Heidelberg

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