RESUMO
BACKGROUND: Limited accessibility of the tumour precludes longitudinal characterisation for therapy guidance in pancreatic ductal adenocarcinoma (PDAC). METHODS: We utilised dielectrophoresis-field flow fractionation (DEP-FFF) to isolate circulating tumour cells (CTCs) in 272 blood draws from 74 PDAC patients (41 localised, 33 metastatic) to non-invasively monitor disease progression. RESULTS: Analysis using multiplex imaging flow cytometry revealed four distinct sub-populations of CTCs: epithelial (E-CTC), mesenchymal (M-CTC), partial epithelial-mesenchymal transition (pEMT-CTC) and stem cell-like (SC-CTC). Overall, CTC detection rate was 76.8% (209/272 draws) and total CTC counts did not correlate with any clinicopathological variables. However, the proportion of pEMT-CTCs (prop-pEMT) was correlated with advanced disease, worse progression-free and overall survival in all patients, and earlier recurrence after resection. CONCLUSION: Our results underscore the importance of immunophenotyping and quantifying specific CTC sub-populations in PDAC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Transição Epitelial-Mesenquimal/fisiologia , Células Neoplásicas Circulantes/patologia , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Farmacológicos/análise , Biomarcadores Farmacológicos/sangue , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/diagnóstico , Células Cultivadas , Progressão da Doença , Monitoramento de Medicamentos/métodos , Feminino , Humanos , Imunofenotipagem , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/classificação , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/diagnóstico , Fenótipo , Valor Preditivo dos Testes , PrognósticoRESUMO
Although dielectrophoresis (DEP) has great potential for addressing clinical cell isolation problems based on cell dielectric differences, a biological basis for predicting the DEP behavior of cells has been lacking. Here, the dielectric properties of the NCI-60 panel of tumor cell types have been measured by dielectrophoretic (DEP) field-flow fractionation, correlated with the exterior morphologies of the cells during growth, and compared with the dielectric and morphological characteristics of the subpopulations of peripheral blood. In agreement with earlier findings, cell total capacitance varied with both cell size and plasma membrane folding and the dielectric properties of the NCI-60 cell types in suspension reflected the plasma membrane area and volume of the cells at their growth sites. Therefore, the behavior of cells in DEP-based manipulations is largely determined by their exterior morphological characteristics prior to release into suspension. As a consequence, DEP is able to discriminate between cells of similar size having different morphological origins, offering a significant advantage over size-based filtering for isolating circulating tumor cells, for example. The findings provide a framework for anticipating cell dielectric behavior on the basis of structure-function relationships and suggest that DEP should be widely applicable as a surface marker-independent method for sorting cells.
Assuntos
Separação Celular/métodos , Eletroforese/métodos , Fracionamento por Campo e Fluxo/métodos , Linhagem Celular Tumoral , Membrana Celular/química , Simulação por Computador , Humanos , Neoplasias/sangue , Neoplasias/química , Neoplasias/patologia , Células Neoplásicas Circulantes/química , Células Neoplásicas Circulantes/patologia , Especificidade de ÓrgãosRESUMO
The scale-invariant property of the cytoplasmic membrane of biological cells is examined by applying the Minkowski-Bouligand method to digitized scanning electron microscopy images of the cell surface. The membrane is found to exhibit fractal behavior, and the derived fractal dimension gives a good description of its morphological complexity. Furthermore, we found that this fractal dimension correlates well with the specific membrane dielectric capacitance derived from the electrorotation measurements. Based on these findings, we propose a new fractal single-shell model to describe the dielectrics of mammalian cells, and compare it with the conventional single-shell model (SSM). We found that while both models fit with experimental data well, the new model is able to eliminate the discrepancy between the measured dielectric property of cells and that predicted by the SSM.
Assuntos
Membrana Celular/química , Membrana Celular/fisiologia , Capacitância Elétrica , Fractais , Modelos Biológicos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Eletroforese , Genisteína/farmacologia , Humanos , RotaçãoRESUMO
Dielectrophoretic field-flow fractionation (DEP-FFF) has been used to discriminate between particles and cells based on their dielectric and density properties. However, hydrodynamic lift forces (HDLF) at flow rates needed for rapid separations were not accounted for in the previous theoretical treatment of the approach. Furthermore, no method was developed to isolate particle or cell physical characteristics directly from DEP-FFF elution data. An extended theory of DEP-FFF is presented that accounts for HDLF. With the use of DS19 erythroleukemia cells as model particles with frequency-dependent dielectric properties, it is shown that the revised theory accounts for DEP-FFF elution behavior over a wide range of conditions and is consistent with sedimentation-FFF when the DEP force is zero. Conducting four elution runs under specified conditions, the theory allows for the derivation of the cell density distribution and provides good estimates of the distributions of the dielectric properties of the cells and their deformability characteristics that affect HDLF. The approach allows for rapid profiling of the biophysical properties of cells, the identification and characterization of subpopulations, and the design of optimal DEP-FFF separation conditions. The extended DEP-FFF theory is widely applicable, and the parameter measurement methods may be adapted easily to other types of particles.
Assuntos
Separação Celular/métodos , Fracionamento por Campo e Fluxo/métodos , Algoritmos , Animais , Fenômenos Biofísicos , Linhagem Celular Tumoral , CamundongosRESUMO
The application of dielectrophoretic field-flow fractionation (depFFF) to the isolation of circulating tumor cells (CTCs) from clinical blood specimens was studied using simulated cell mixtures of three different cultured tumor cell types with peripheral blood. The depFFF method can not only exploit intrinsic tumor cell properties so that labeling is unnecessary but can also deliver unmodified, viable tumor cells for culture and/or all types of molecular analysis. We investigated tumor cell recovery efficiency as a function of cell loading for a 25 mm wide x 300 mm long depFFF chamber. More than 90% of tumor cells were recovered for small samples but a larger chamber will be required if similarly high recovery efficiencies are to be realized for 10 mL blood specimens used CTC analysis in clinics. We show that the factor limiting isolation efficiency is cell-cell dielectric interactions and that isolation protocols should be completed within approximately 15 min in order to avoid changes in cell dielectric properties associated with ion leakage.
Assuntos
Separação Celular/métodos , Eletroforese , Fracionamento por Campo e Fluxo , Células Neoplásicas Circulantes , Linhagem Celular Tumoral , Desenho de Equipamento , Feminino , Humanos , Leucócitos MononuclearesRESUMO
We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations.
Assuntos
Tecido Adiposo , Separação Celular/métodos , Eletroforese/instrumentação , Eletroforese/métodos , Fracionamento por Campo e Fluxo/instrumentação , Fracionamento por Campo e Fluxo/métodos , Células-Tronco , HumanosRESUMO
Dielectrophoretic field-flow fractionation (dFFF) was applied as a contact-free way to sense changes in the plasma membrane capacitances and conductivities of cultured human HL-60 cells in response to toxicant exposure. A micropatterned electrode imposed electric forces on cells in suspension in a parabolic flow profile as they moved through a thin chamber. Relative changes in the dFFF peak elution time, reflecting changes in cell membrane area and ion permeability, were measured as indices of response during the first 150 min of exposure to eight toxicants having different single or mixed modes of action (acrylonitrile, actinomycin D, carbon tetrachloride, endosulfan, N-nitroso- N-methylurea (NMU), paraquat dichloride, puromycin, and styrene oxide). The dFFF method was compared with the cell viability assay for all toxicants and with the mitochondrial potentiometric dye assay or DNA alkaline comet assay according to the mode of action of the specific agents. Except for low doses of nucleic acid-targeting agents (actinomycin D and NMU), the dFFF method detected all toxicants more sensitively than other assays, in some cases up to 10 (5) times more sensitively than the viability approach. The results suggest the dFFF method merits additional study for possible applicability in toxicology.
Assuntos
Fracionamento por Campo e Fluxo/métodos , Poluentes Químicos da Água/análise , Linhagem Celular Tumoral , Células/efeitos dos fármacos , Células/metabolismo , Impedância Elétrica , Humanos , Sensibilidade e Especificidade , Poluentes Químicos da Água/toxicidadeRESUMO
As an approach to isolating tumor cells from fine needle biopsy specimens, we investigated a dielectric cell preparation method using an in vivo xenographic tumor model. Cultured human MDA-MB-435 tumor cells were grown as solid tumors in nude mice and fine needle aspiration biopsies were conducted. Biopsied cells were suspended in sucrose medium and collected on slides patterned with microelectrode arrays (electrosmears) energized by electrical signals in the range 10 to 960 kHz. The unlabeled cells adhered to characteristic regions of the slides in accordance with their morphology as a result of dielectric forces. Tumor cells were trapped between 40 and 60 kHz and were separated according to whether they were mitotic, large and complex, or small. Damaged tumor cells were captured at between 60 and 120 kHz; granulocytes between 70 and 90 kHz; lymphocytes between 85 and 105 kHz; healthy erythrocytes between 140 and 180 kHz, and damaged erythrocytes above 180 kHz. Using intrinsic cell characteristics, the electrosmear presented cell subpopulations from fine needle aspiration biopsy specimens in a manner that is compatible with automated slide-based analysis systems. The approach has the potential to facilitate the analysis of the role of cell subpopulations in disease.
Assuntos
Biópsia por Agulha Fina , Separação Celular , Eletricidade , Neoplasias/patologia , Transplante Heterólogo , Animais , Linhagem Celular Tumoral , Separação Celular/instrumentação , Separação Celular/métodos , Humanos , Camundongos , Camundongos NusRESUMO
This paper presents a continuous-flow polymerase chain reaction (PCR) microchip with a serpentine microchannel of varying width for "regional velocity control." Varying the channel width by incorporating expanding and contracting conduits made it possible to control DNA sample velocities for the optimization of the exposure times of the sample to each temperature phase while minimizing the transitional periods during temperature transitions. A finite element analysis (FEA) and semi-analytical heat transfer model was used to determine the distances between the three heating assemblies that are responsible for creating the denaturation (96 degrees C), hybridization (60 degrees C), and extension (72 degrees C) temperature zones within the microchip. Predictions from the thermal FEA and semi-analytical model were compared with temperature measurements obtained from an infrared (IR) camera. Flow-field FEAs were also performed to predict the velocity distributions in the regions of the expanding and contracting conduits to study the effects of the microchannel geometry on flow recirculation and bubble nucleation. The flow fields were empirically studied using micro particle image velocimetry (mu-PIV) to validate the flow-field FEA's and to determine experimental velocities in each of the regions of different width. Successful amplification of a 90 base pair (bp) bacillus anthracis DNA fragment was achieved.
RESUMO
The dielectrophoretic (DEP) crossover method has been applied to the detection of cell responses to toxicants. Time and dose responses of the human cultured leukemia (HL-60) line were measured for paraquat, styrene oxide (SO), N-nitroso-N-methylurea (NMU) and puromycin. These toxicants were chosen because of their different predominant mechanisms of action, namely membrane free radical attack, simultaneous membrane and nucleic acid attack, nucleic acid alkylation, and protein synthesis inhibition, respectively. For all treatments, the specific membrane capacitance (C(mem)) of the cells decreased while the specific membrane conductance (G(mem)) increased in dose- and time-dependent manners. The DEP responses correlated sensitively with alterations in cell surface morphology, especially folds, microvilli, and blebs, observed by scanning electron microscopy. The DEP method was more sensitive to agents that had a direct action on the membrane than to agents for which membrane alterations were secondary. The responses to paraquat and SO, which directly damaged the cell membrane, could be detected 15 min after exposure, while those for puromycin and NMU, which acted on intracellular targets, could be detected after 30 min. The detection times and dose sensitivity results showed that the DEP method is much faster and more sensitive than conventional cell and higher organism viability testing techniques. The feasibility of producing small instruments for toxicity detection and screening based on cellular dielectric responses is discussed.
Assuntos
Membrana Celular/efeitos dos fármacos , Eletroforese/métodos , Toxicologia/métodos , Membrana Celular/química , Membrana Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Capacitância Elétrica , Condutividade Elétrica , Eletroforese/instrumentação , Compostos de Epóxi/toxicidade , Citometria de Fluxo , Células HL-60 , Humanos , Metilnitrosoureia/toxicidade , Microquímica/métodos , Microscopia Eletrônica de Varredura , Paraquat/toxicidade , Puromicina/toxicidade , Propriedades de Superfície , Fatores de TempoRESUMO
The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6+/-0.9 to 9.1+/-0.5 mF/m(2) 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.
Assuntos
Apoptose , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Separação Celular/métodos , Fragmentação do DNA , Capacitância Elétrica , Condutividade Elétrica , Eletroforese/métodos , Citometria de Fluxo , Genisteína/farmacologia , Inibidores do Crescimento/farmacologia , Células HL-60 , Humanos , Microscopia Eletrônica de Varredura , Necrose , Fosfatidilserinas/metabolismo , Fatores de TempoRESUMO
PURPOSE: Electrorotation (ROT) is a technique that allows for determination of the dielectric properties of living cells when exposed to a rotating electric field. We evaluated the ROT behavior of MCF/neo and p185(neu) transfectancts MCF/HER2-11 and MCF/HER2-18 to investigate whether differences in HER-2/neu expression were associated with differences in dielectric properties in these cells. EXPERIMENTAL DESIGN: P185(neu) was measured by Western blotting in MCF/neo cells and HER-2/neu transfectants MCF/HER2-11 and MCF/HER2-18. ROT spectra and cell membrane-specific capacitance were obtained for each cell line. RESULTS: The mean cell membrane-specific capacitance values for MCF/neo, MCF/HER2-11, and MCF/HER2-18 were 2.09, 1.70, and 2.56 microF/cm(2), respectively. The mean specific capacitance for MCF/neo was significantly different from that for MCF/HER2-11 (P = 0.006) and that for MCF/HER2-18 (P = 0.007). CONCLUSIONS: ROT is sufficiently sensitive to detect variations in dielectric properties in breast cancer cell lines overexpressing p185(neu). These differences may be related to the morphological alterations determined by HER-2/neu overexpression.
Assuntos
Biofísica/métodos , Neoplasias da Mama/metabolismo , Condutividade Elétrica , Receptor ErbB-2/biossíntese , Automação , Western Blotting , Humanos , Sensibilidade e Especificidade , Transfecção , Células Tumorais CultivadasRESUMO
We describe the manipulation of aqueous droplets in an immiscible, low-permittivity suspending medium. Such droplets may serve as carriers for not only air- and water-borne samples, contaminants, chemical reagents, viral and gene products, and cells, but also the reagents to process and characterise these samples. We present proofs-of-concept for droplet manipulation through dielectrophoresis by: (1). moving droplets on a two-dimensional array of electrodes, (2). achieving dielectrically-activated droplet injection, (3). fusing and reacting droplets, and (4). conducting a basic biological assay through a combination of these steps. A long-term goal of this research is to provide a platform fluidic processor technology that can form the core of versatile, automated, micro-scale devices to perform chemical and biological assays at or near the point of care, which will increase the availability of modern medicine to people who do not have ready access to modern medical institutions, and decrease the cost and delays associated with that lack of access.
Assuntos
Eletroforese/métodos , Microfluídica/métodos , Eletrodos , Fluorescência , Nanotecnologia , Soroalbumina Bovina/análise , Software , Água , o-FtalaldeídoRESUMO
An important enabling factor for realising integrated micro fluidic analysis instruments for medical diagnostics purposes is front-end sample preparation. Dielectrophoresis is a method that offers great potential for cell discrimination and isolation for sample processing, and here we have applied it to the problem of isolating malaria-infected cells from blood. During development of the malarial pathogen, Plasmodium falciparum, increases occur in the ionic permeability of the plasma membrane of infected erythrocytes. When challenged by suspension in a low conductivity medium, infected cells lose internal ions while uninfected cells retain them. The resultant dielectric differences between infected and uninfected cells were exploited by dielectrophoretic manipulation in spatially inhomogeneous, travelling electrical fields produced by two types of microelectrode arrays. Parasitised cells of ring form or later stage from cultures and clinical specimens were isolated by steric dielectric field-flow-fractionation, focused at the centre of a spiral electrode array, and identified and counted. The dielectrophoretic methods require only a few micro litres of blood, and should be applicable to the production of small, low-cost automated devices for assessing parasite concentrations with potential applicability to drug sensitivity studies and the diagnosis of malaria. By simple adjustment of the electrical field parameters, other cell subpopulations that characterise disease, such as residual cancer cells in blood, can be similarly isolated and analysed.
Assuntos
Eletroforese/métodos , Eritrócitos/parasitologia , Plasmodium falciparum/isolamento & purificação , Animais , MicroeletrodosRESUMO
Droplet-based programmable processors promise to offer solutions to a wide range of applications in which chemical and biological analysis and/or small-scale synthesis are required, suggesting they will become the microfluidic equivalents of microprocessors by offering off-the-shelf solutions for almost any fluid based analysis or small scale synthesis problem. A general purpose droplet processor should be able to manipulate droplets of different compositions (including those that are electrically conductive or insulating and those of polar or non-polar nature), to control reagent titrations accurately, and to remain free of contamination and carry over on its reaction surfaces. In this article we discuss the application of dielectrophoresis to droplet based processors and demonstrate that it can provide the means for accurately titrating, moving and mixing polar or non-polar droplets whether they are electrically conductive or not. DEP does not require contact with control surfaces and several strategies for minimizing surface contact are presented. As an example of a DEP actuated general purpose droplet processor, we show an embodiment based on a scaleable CMOS architecture that uses DEP manipulation on a 32 x 32 electrode array having built-in control and switching circuitry. Lastly, we demonstrate the concept of a general-purpose programming environment that facilitates droplet software development for any type of droplet processor.
Assuntos
Eletroforese/métodos , Processamento de Imagem Assistida por Computador , Microfluídica , Eletroforese/instrumentação , Microfluídica/instrumentação , Microfluídica/métodos , Movimento (Física) , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
We have used self-assembled monolayer techniques to produce a new class of microspheres with specifically engineered dielectric properties to enable their dielectrophoretic manipulation and identification in microsystems. Dielectrophoresis is an electrokinetic phenomenon that exploits frequency-dependent polarizability differences between a particle and its suspending medium to drive the movement of the particle toward or away from the high-field regions of an inhomogeneous electric field. While dielectrophoretic methods have been used extensively for cell manipulation, separation, and identification, we wished to extend the applicability of dielectrophoresis to molecular analysis by developing a panel of dielectric microspheres or "handles". Dielectric shell theory was used to model the dielectrophoretic response for a biomimetic particle composed of a thin insulating shell over a conductive interior. We specifically sought to modulate the specific capacitance, and thereby the dielectric properties, of the particle by controlling the thickness of the insulating layer. Such a structure was fabricated by covering a gold-coated polystyrene core particle with self-assembled monolayers of alkanethiol and phospholipid. To test the prediction that the carbon chain length of these layers should dictate the dielectric properties of the particles, we constructed a panel of six microsphere types with shell compositions ranging from a C(9) alkanethiol monolayer to a C(32) hybrid bilayer membrane. These microsphere populations were distinguishable and manipulatable by dielectrophoresis in a characteristic, frequency-dependent manner as predicted by theory. Experimentally derived specific membrane capacitance values were inversely related to the insulating shell thickness and agreed with published capacitance values for planar layers of similar thicknesses. These proof of principle studies are the first to demonstrate that the dielectric properties of particles can be specifically engineered to allow their dielectrophoretic manipulation and are a first step toward the development of bead-based dielectrophoretic microsystems for multiplexed molecular separation and analysis.
RESUMO
Microfluidic systems are under development to address a variety of medical problems. Key advantages of micrototal analysis systems based on microfluidic technology are the promise of small size and the integration of sample handling and measurement functions within a single, automated device having low mass-production costs. Here, we review the spectrum of methods currently used to detect malaria, consider their advantages and disadvantages, and discuss their adaptability towards integration into small, automated micro total analysis systems. Molecular amplification methods emerge as leading candidates for chip-based systems because they offer extremely high sensitivity, the ability to recognize malaria species and strain, and they will be adaptable to the detection of new genotypic signatures that will emerge from current genomic-based research of the disease. Current approaches to the development of chip-based molecular amplification are considered with special emphasis on flow-through PCR, and we present for the first time the method of malaria specimen preparation by dielectrophoretic field-flow-fractionation. Although many challenges must be addressed to realize a micrototal analysis system for malaria diagnosis, it is concluded that the potential benefits of the approach are well worth pursuing.
Assuntos
Malária/diagnóstico , Microfluídica/métodos , Avaliação da Tecnologia Biomédica/métodos , Eletroforese/métodos , Humanos , Reação em Cadeia da Polimerase , Avaliação da Tecnologia Biomédica/tendênciasRESUMO
As the molecular origins of disease are better understood, the need for affordable, rapid, and automated technologies that enable microscale molecular diagnostics has become apparent. Widespread use of microsystems that perform sample preparation and molecular analysis could ensure that the benefits of new biomedical discoveries are realized by a maximum number of people, even those in environments lacking any infrastructure. While progress has been made in developing miniaturized diagnostic systems, samples are generally processed off-device using labor-intensive and time-consuming traditional sample preparation methods. We present the concept of an integrated programmable general-purpose sample analysis processor (GSAP) architecture where raw samples are routed to separation and analysis functional blocks contained within a single device. Several dielectrophoresis-based methods that could serve as the foundation for building GSAP functional blocks are reviewed including methods for cell and particle sorting, cell focusing, cell ac impedance analysis, cell lysis, and the manipulation of molecules and reagent droplets.
RESUMO
Recent measurements have demonstrated that the dielectric properties of cells depend on their type and physiological status. For example, MDA-231 human breast cancer cells were found to have a mean plasma membrane specific capacitance of 26 mF/m(2), more than double the value (11 mF/m(2)) observed for resting T-lymphocytes. When an inhomogeneous ac electric field is applied to a particle, a dielectrophoretic (DEP) force arises that depends on the particle dielectric properties. Therefore, cells having different dielectric characteristics will experience differential DEP forces when subjected to such a field. In this article, we demonstrate the use of differential DEP forces for the separation of several different cancerous cell types from blood in a dielectric affinity column. These separations were accomplished using thin, flat chambers having microelectrode arrays on the bottom wall. DEP forces generated by the application of ac fields to the electrodes were used to influence the rate of elution of cells from the chamber by hydrodynamic forces within a parabolic fluid flow profile. Electrorotation measurements were first made on the various cell types found within cell mixtures to be separated, and theoretical modeling was used to derive the cell dielectric parameters. Optimum separation conditions were then predicted from the frequency and suspension conductivity dependencies of cell DEP responses defined by these parameters. Cell separations were then undertaken for various ratios of cancerous to normal cells at different concentrations. Eluted cells were characterized in terms of separation efficiency, cell viability, and separation speed. For example, 100% efficiency was achieved for purging MDA-231 cells from blood at the tumor to normal cell ratio 1:1 x 10(5) or 1:3 x 10(5), cell viability was not compromised, and separation rates were at least 10(3) cells/s. Theoretical and experimental criteria for the design and operation of such separators are presented.