Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients.

BACKGROUND: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. METHODS: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. RESULTS: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. CONCLUSION: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Fast Microscale Acoustic Streaming Driven by a Temperature-Gradient-Induced Nondissipative Acoustic Body Force.

We study acoustic streaming in liquids driven by a nondissipative acoustic body force created by light-induced temperature gradients. This thermoacoustic streaming produces a velocity amplitude nearly 100 times higher than the boundary-driven Rayleigh streaming and the Rayleigh-Bénard convection at a temperature gradient of 10 K/mm in the channel. The Rayleigh streaming is altered by the acoustic body force at a temperature gradient of only 0.5 K/mm. The thermoacoustic streaming allows for modular flow control and enhanced heat transfer at the microscale. Our study provides the groundwork for studying microscale acoustic streaming coupled with temperature fields.

Acoustic Streaming and Its Suppression in Inhomogeneous Fluids.

We present a theoretical and experimental study of boundary-driven acoustic streaming in an inhomogeneous fluid with variations in density and compressibility. In a homogeneous fluid this streaming results from dissipation in the boundary layers (Rayleigh streaming). We show that in an inhomogeneous fluid, an additional nondissipative force density acts on the fluid to stabilize particular inhomogeneity configurations, which markedly alters and even suppresses the streaming flows. Our theoretical and numerical analysis of the phenomenon is supported by ultrasound experiments performed with inhomogeneous aqueous iodixanol solutions in a glass-silicon microchip.

Clinical-Scale Cell-Surface-Marker Independent Acoustic Microfluidic Enrichment of Tumor Cells from Blood.

Enumeration of circulating tumor cells (CTCs) predicts overall survival and treatment response in metastatic cancer, but as many commercialized assays isolate CTCs positive for epithelial cell markers alone, CTCs with little or no epithelial cell adhesion molecule (EpCAM) expression stay undetected. Therefore, CTC enrichment and isolation by label-free methods based on biophysical rather than biochemical properties could provide a more representative spectrum of CTCs. Here, we report on a clinical-scale automated acoustic microfluidic platform processing 5 mL of erythrocyte-depleted paraformaldehyde (PFA)-fixed blood (diluted 1:2) at a flow rate of 75 µL/min, recovering 43/50 (86 ± 2.3%) breast cancer cell line cells (MCF7), with 0.11% cancer cell purity and 162-fold enrichment in close to 2 h based on intrinsic biophysical cell properties. Adjustments of the voltage settings aimed at higher cancer cell purity in the central outlet provided 0.72% cancer cell purity and 1445-fold enrichment that resulted in 62 ± 8.7% cancer cell recovery. Similar rates of cancer-cell recovery, cancer-cell purity, and fold-enrichment were seen with both prostate cancer (DU145, PC3) and breast cancer (MCF7) cell line cells. We identified eosinophil granulocytes as the predominant white blood cell (WBC) contaminant (85%) in the enriched cancer-cell fraction. Processing of viable cancer cells in erythrocyte-depleted blood provided slightly reduced results as to fixed cells (77% cancer cells in the enriched cancer cell fraction, with 0.2% WBC contamination). We demonstrate feasibility of enriching either PFA-fixed or viable cancer cells with a clinical-scale acoustic microfluidic platform that can be adjusted to meet requirements for either high cancer-cell recovery or higher purity and can process 5 mL blood samples in close to 2 h.

Acoustic Force Density Acting on Inhomogeneous Fluids in Acoustic Fields.

We present a theory for the acoustic force density acting on inhomogeneous fluids in acoustic fields on time scales that are slow compared to the acoustic oscillation period. The acoustic force density depends on gradients in the density and compressibility of the fluid. For microfluidic systems, the theory predicts a relocation of the inhomogeneities into stable field-dependent configurations, which are qualitatively different from the horizontally layered configurations due to gravity. Experimental validation is obtained by confocal imaging of aqueous solutions in a glass-silicon microchip.

Acoustofluidic, label-free separation and simultaneous concentration of rare tumor cells from white blood cells.

Enrichment of rare cells from peripheral blood has emerged as a means to enable noninvasive diagnostics and development of personalized drugs, commonly associated with a prerequisite to concentrate the enriched rare cell population prior to molecular analysis or culture. However, common concentration by centrifugation has important limitations when processing low cell numbers. Here, we report on an integrated acoustophoresis-based rare cell enrichment system combined with integrated concentration. Polystyrene 7 µm microparticles could be separated from 5 µm particles with a recovery of 99.3 ± 0.3% at a contamination of 0.1 ± 0.03%, with an overall 25.7 ± 1.7-fold concentration of the recovered 7 µm particles. At a flow rate of 100 µL/min, breast cancer cells (MCF7) spiked into red blood cell-lysed human blood were separated with an efficiency of 91.8 ± 1.0% with a contamination of 0.6 ± 0.1% from white blood cells with a 23.8 ± 1.3-fold concentration of cancer cells. The recovery of prostate cancer cells (DU145) spiked into whole blood was 84.1 ± 2.1% with 0.2 ± 0.04% contamination of white blood cells with a 9.6 ± 0.4-fold concentration of cancer cells. This simultaneous on-chip separation and concentration shows feasibility of future acoustofluidic systems for rapid label-free enrichment and molecular characterization of circulating tumor cells using peripheral venous blood in clinical practice.

Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing.

Efficient techniques for separating target cells from undiluted blood are necessary for various diagnostic and research applications. This paper presents acoustic focusing in dense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from whole blood in a label-free and flow-through format. If the blood is laminated or mixed with iodixanol solutions while passing through the resonant microchannel, all the components (fluids and cells) rearrange according to their acoustic impedances. Red blood cells (RBCs) have higher effective acoustic impedance than PBMCs. Therefore, they relocate to the pressure node despite the dense medium, while PBMCs stay near the channel walls due to their negative contrast factor relative to their surrounding medium. By modifying the medium and thus tuning the contrast factor of the cells, we enriched PBMCs relative to RBCs by a factor of 3600 to 11,000 and with a separation efficiency of 85%. That level of RBC depletion is higher than most other microfluidic methods and similar to that of density gradient centrifugation. The current acoustophoretic chip runs up to 20 µl/min undiluted whole blood and can be integrated with downstream analysis.

Acoustophoretic Characterization and Separation of Blood Cells in Acoustic Impedance Gradients.

Single-cell phenotyping based on biophysical properties is a promising tool to distinguish cell types and their response to a given condition, and charting such properties also enables optimization of cell separations. Isoacoustic focusing, where cells migrate to their points of zero acoustic contrast in an acoustic impedance gradient, added the effective acoustic impedance of cells to the directory of biophysical properties that can be utilized to categorize or separate cells. This study investigates isoacoustic focusing in a stop-flow regime and shows how cells migrate towards their isoacoustic point. We introduce a numerical model that we use to estimate the acoustic energy density in acoustic impedance gradient media by tracking particles of known properties, and we investigate the effect of acoustic streaming. From the measured trajectories of cells combined with fluorescence intensity images of the slowly diffusing gradient, we read out the effective acoustic impedance of neutrophils and K562 cancer cells. Finally, we propose suitable acoustic impedance gradients that lead to a high degree separation of neutrophils and K562 cells in a continuous-flow configuration.

Acoustic enrichment of heterogenous circulating tumor cells and clusters from patients with metastatic prostate cancer.

Background: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Methods: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility) resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Results: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogenous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC-clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding higher number of CTCs using acoustophoresis. Conclusion: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC-clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables sensitive label-free enrichment of cells with epithelial phenotype in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Microfluidic, label-free enrichment of prostate cancer cells in blood based on acoustophoresis.

Circulating tumor cells (CTC) are shed in peripheral blood at advanced metastatic stages of solid cancers. Surface-marker-based detection of CTC predicts recurrence and survival in colorectal, breast, and prostate cancer. However, scarcity and variation in size, morphology, expression profile, and antigen exposure impairs reliable detection and characterization of CTC. We have developed a noncontact, label-free microfluidic acoustophoresis method to separate prostate cancer cells from white blood cells (WBC) through forces generated by ultrasonic resonances in microfluidic channels. Implementation of cell prealignment in a temperature-stabilized (±0.5 °C) acoustophoresis microchannel dramatically enhanced the discriminatory capacity and enabled the separation of 5 µm microspheres from 7 µm microspheres with 99% purity. Next, we determined the feasibility of employing label-free microfluidic acoustophoresis to discriminate and divert tumor cells from WBCs using erythrocyte-lysed blood from healthy volunteers spiked with tumor cells from three prostate cancer cell-lines (DU145, PC3, LNCaP). For cells fixed with paraformaldehyde, cancer cell recovery ranged from 93.6% to 97.9% with purity ranging from 97.4% to 98.4%. There was no detectable loss of cell viability or cell proliferation subsequent to the exposure of viable tumor cells to acoustophoresis. For nonfixed, viable cells, tumor cell recovery ranged from 72.5% to 93.9% with purity ranging from 79.6% to 99.7%. These data contribute proof-in-principle that label-free microfluidic acoustophoresis can be used to enrich both viable and fixed cancer cells from WBCs with very high recovery and purity.

Label-free somatic cell cytometry in raw milk using acoustophoresis.

A microfluidic system for cell enumeration in raw milk was developed. The new method, preconditions the milk sample using acoustophoresis that removes lipid particles which are larger than a few micrometers. The acoustophoretic preprocessing eliminates the need for conventional sample preparation techniques, which include chemical solvents, cell labeling and centrifugation, and facilitates rapid cell enumeration using microscopy or coulter counter measurements. By introducing an acoustic standing wave with three pressure nodes in a microchannel at the same time as the milk sample is laminated to the channel center, lipids are acoustically driven to the closest pressure antinode at each side of the channel center and the cells in the milk sample are focused in the central pressure node. The extracted center fraction with cells becomes sufficiently clean from lipid vesicles to enable enumeration of somatic cells without any labeling step either by direct light microscopy or by coulter counting. Obtained lipid free milk fractions clearly revealed the cell fraction when analyzed by Coulter Counting. Cell counting as measured by a Coulter Counter after acoustophoretic lipid depletion aligned with the corresponding data obtained by reference measurements based on fluorescence staining and subsequent flow cytometer analysis.

Measuring the local pressure amplitude in microchannel acoustophoresis.

A new method is reported on how to measure the local pressure amplitude and the Q factor of ultrasound resonances in microfluidic chips designed for acoustophoresis of particle suspensions. The method relies on tracking individual polystyrene tracer microbeads in straight water-filled silicon/glass microchannels. The system is actuated by a PZT piezo transducer attached beneath the chip and driven by an applied ac voltage near its eigenfrequency of 2 MHz. For a given frequency a number of particle tracks are recorded by a CCD camera and fitted to a theoretical expression for the acoustophoretic motion of the microbeads. From the curve fits we obtain the acoustic energy density, and hence the pressure amplitude as well as the acoustophoretic force. By plotting the obtained energy densities as a function of applied frequency, we obtain Lorentzian line shapes, from which the resonance frequency and the Q factor for each resonance peak are derived. Typical measurements yield acoustic energy densities of the order of 10 J/m(3), pressure amplitudes of 0.2 MPa, and Q factors around 500. The observed half wavelength of the transverse acoustic pressure wave is equal within 2% to the measured width w = 377 microm of the channel.

Particle-size-dependent acoustophoretic motion and depletion of micro- and nano-particles at long timescales.

We present three-dimensional measurements of particle-size-dependent acoustophoretic motion of microparticles with diameters from 4.8 µm down to 0.5 µm suspended in either homogeneous or inhomogeneous fluids inside a glass-silicon microchannel and exposed to a standing ultrasound wave. To study the crossover from radiation force dominated to streaming dominated motion as the particle size is decreased, we extend previous studies to long timescales, where the particles smaller than the crossover size move over distances comparable to the channel width. We observe a particle-size-dependent particle depletion at late times for the particles smaller than the crossover size. The mechanisms behind this depletion in homogeneous fluids are rationalized by numerical simulations which take the Brownian motion into account. Experimentally, the particle trajectories in inhomogeneous fluids show focusing in the bulk of the microchannel at early times, even for the particles below the critical size, which clearly demonstrates the potential to manipulate submicrometer particles.

Gradient acoustic focusing of sub-micron particles for separation of bacteria from blood lysate.

Handling of submicron-sized objects is important in many biochemical and biomedical applications, but few methods today can precisely manipulate this range of particles. We present gradient acoustic focusing that enables flow-through particle separation of submicron particles and cells and we apply it for separation of bacteria from blood lysate to facilitate their detection in whole blood for improved diagnostics. To control suspended objects below the classical 2µm size limit for acoustic focusing, we introduce a co-flowing acoustic impedance gradient to generate a stabilizing acoustic volume force that supresses acoustic streaming. The method is validated theoretically and experimentally using polystyrene particles, Staphylococcus aureus, Streptococcus pneumoniae and Escherichia coli. The applicability of the method is demonstrated by the separation of bacteria from selectively chemically lysed blood. Combined with downstream operations, this new approach opens up for novel methods for sepsis diagnostics.

Decomplexing biofluids using microchip based acoustophoresis.

Highly efficient washing and extraction of microbeads to decomplex analytes ranging from small peptides to large viruses was realised in a microscaled continuous flow format. The bead washing principle reported herein is based on acoustophoresis, i.e. the primary acoustic radiation force in an ultrasonic standing wave and laminar flow properties are utilised to translate bioanalytes trapped on functionalised microbeads from one carrier fluid to another. The carry-over of non-specific material ranges from 1 to 50 ppm relative to input levels depending on application, making acoustophoresis suitable for extraction of rare species from complex environments. Selective extraction of a phosphopeptide relative to its unphosphorylated counterpart is demonstrated using metal oxide affinity capture (MOAC) beads and MALDI-TOF MS readout. Acoustophoresis of microbeads activated with specific binders could be used to capture phage viral particles. The efficiency of the acoustophoretic washing principle was demonstrated by an unspecific phage cross contamination level of only 10(-6) of that in the input bead/phage mixture. The continuous flow format makes acoustophoretic washing flexible regarding sample volume and also allows for easy integration into a sequence of particle handling and analytical unit operations.

Harmonic microchip acoustophoresis: a route to online raw milk sample precondition in protein and lipid content quality control.

A microfluidic approach for raw milk sample preconditioning prior to protein and lipid content analysis has been developed. The system utilizes microchip acoustophoresis and is a further extension of our previously reported multiple node ultrasonic standing wave focusing platform (Grenvall, C., Augustsson, P., Matsuoka, H. and Laurell, T. Proc. Micro Total Anal. Syst. 2008, 1, 161-163). The microfluidic approach offers a method for rapid raw milk quality control using Fourier transform infrared spectroscopy (FT-IR). Two acoustophoresis modes are explored, 2 lambda/2 and 3 lambda/2, offering lipid content enrichment or depletion, respectively. Lipid content depletion above 90% was accomplished. FT-IR data on microchip-processed raw milk samples, enabling direct lipid and protein content analysis, are reported. Most importantly, the harmonic operational modes bypass the problem of lipid aggregation and subsequent clogging, inherent in lambda/2 acoustophoresis systems.

Acoustic impedance matched buffers enable separation of bacteria from blood cells at high cell concentrations.

Sepsis is a common and often deadly systemic response to an infection, usually caused by bacteria. The gold standard for finding the causing pathogen in a blood sample is blood culture, which may take hours to days. Shortening the time to diagnosis would significantly reduce mortality. To replace the time-consuming blood culture we are developing a method to directly separate bacteria from red and white blood cells to enable faster bacteria identification. The blood cells are moved from the sample flow into a parallel stream using acoustophoresis. Due to their smaller size, the bacteria are not affected by the acoustic field and therefore remain in the blood plasma flow and can be directed to a separate outlet. When optimizing for sample throughput, 1 ml of undiluted whole blood equivalent can be processed within 12.5 min, while maintaining the bacteria recovery at 90% and the blood cell removal above 99%. That makes this the fastest label-free microfluidic continuous flow method per channel to separate bacteria from blood with high bacteria recovery (>80%). The high throughput was achieved by matching the acoustic impedance of the parallel stream to that of the blood sample, to avoid that acoustic forces relocate the fluid streams.

Acoustofluidic hematocrit determination.

Hematocrit (HCT) measurements of blood from patients, blood donors and athletes are routinely performed on a daily basis. These measurements are often performed in centralized hospital labs by whole blood analyzers, which leads to long time-to-result. On site measurements, based on centrifugation can be done, but these assays require manual handling, are slow and can just measure HCT in contrast to the central lab whole blood analyzers. In this work, we present a microfluidic based method to measure HCT in blood samples by acoustic separation of whole blood into discrete regions of plasma and red blood cells. Comparison of the areas of the red blood cell and plasma regions gives an accurate HCT value, with a linear correlation to the centrifugation-based reference method. A readout can be performed within 2 s of acoustic actuation providing a readout accuracy of approximately 3% points (pp) HCT. Additional accuracy can be achieved by extending the acoustic actuation to 20 s, yielding an error of less than 1 pp HCT. This acoustic tool is optimal for integration into a lab-on-a-chip device with in-line measurements of different clinical parameters.

Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping.

Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size.

Label-free concentration of viable neurons, hESCs and cancer cells by means of acoustophoresis.

Concentration of viable cell populations in suspension is of interest for several clinical and pre-clinical applications. Here, we report that microfluidic acoustophoresis is an effective method to efficiently concentrate live and viable cells with high target purity without any need for protein fluorescent labeling using antibodies or over-expression. We explored the effect of the acoustic field acoustic energy density and systematically used different protocols to induce apoptosis or cell death and then determined the efficiency of live and dead cell separation. We used the breast cancer cell line MCF-7, the mouse neuroblastoma N2a as well as human embryonic stem cells (hESCs) to demonstrate that this method is gentle and can be applied to different cell populations. First, we induced cell death by means of high osmotic shock using a high concentration of PBS (10×), the protein kinase inhibitor staurosporine, high concentrations of dimethyl sulfoxide (DMSO, 10%), and finally, cell starvation. In all the methods employed, we successfully induced cell death and were able to purify and concentrate the remaining live cells using acoustophoresis. Importantly, the concentration of viable cells was not dependent on a specific cell type. Further, we demonstrate that different death inducing stimuli have different effects on the intrinsic cell properties and therefore affect the efficiency of the acoustophoretic separation.