We developed a microfluidics-based model to quantify cell-level processes modulating the pathophysiology of sickle cell disease (SCD). This in vitro model enabled quantitative investigations of the kinetics of cell sickling, unsickling, and cell rheology. We created short-term and long-term hypoxic conditions to simulate normal and retarded transit scenarios in microvasculature. Using blood samples from 25 SCD patients with sickle hemoglobin (HbS) levels varying from 64 to 90.1%, we investigated how cell biophysical alterations during blood flow correlated with hematological parameters, HbS level, and hydroxyurea (HU) therapy. From these measurements, we identified two severe cases of SCD that were also independently validated as severe from a genotype-based disease severity classification. These results point to the potential of this method as a diagnostic indicator of disease severity. In addition, we investigated the role of cell density in the kinetics of cell sickling. We observed an effect of HU therapy mainly in relatively dense cell populations, and that the sickled fraction increased with cell density. These results lend support to the possibility that the microfluidic platform developed here offers a unique and quantitative approach to assess the kinetic, rheological, and hematological factors involved in vasoocclusive events associated with SCD and to develop alternative diagnostic tools for disease severity to supplement other methods. Such insights may also lead to a better understanding of the pathogenic basis and mechanism of drug response in SCD.sickle cell anemia | vasoocclusion | capillary obstruction ratio | cell deformability | Aes-103

In previous work, a capillary electrophoresis sodium dodecyl sulfate (CE-SDS) method using precolumn labeling and laser-induced fluorescence (LIF) detection was developed at Genentech Inc. as part of the control system for the quality control release of a recombinant monoclonal antibody (rMAb) (Hunt, G.; Nashabeh, W. Anal. Chem. 1999, 71, 2390-2397.). In the current work, a generic and quantitative CE-SDS assay with LIF detection of rMAbs with improved accuracy and precision is described. The implementation of an alkylating step with iodoacetamide and optimization of the incubation temperature and time, in the presence of SDS, greatly decrease any thermally induced fragmentation of nonreduced labeled rMAb samples. In addition, a quantitative study of the effects of sample buffer pH on rMAb fragmentation is also presented. Furthermore, the performance of alternative CE-SDS polymer solutions and instrumentation for quantitative analysis of rMAbs is shown in this article. The validation of this method, under the guidelines of the International Committee on Harmonization (ICH), demonstrates that the assay quantitatively determines the consistency of rMAb manufacture as it relates to size heterogeneity and product purity.

We present an experimental method to quantitatively characterize the mechanical properties of a large number of biological cells by introducing controlled deformation through dielectrophoresis in a microfluidic device. We demonstrate the capability of this technique by determining the force versus deformation characteristics of healthy human red blood cells (RBCs) and RBCs infected in vitro with Plasmodium falciparum malaria parasites. These experiments clearly distinguish uninfected and healthy RBCs from infected ones, and the mechanical signatures extracted from these tests are in agreement with data from other independent methods. The method developed here thus provides a potentially helpful tool to characterize quickly and effectively the isolated biomechanical response of cells in a large population, for probing the pathological states of cells, disease diagnostics, and drug efficacy assays.

The electrical properties of biological cells have connections to their pathological states. Here we present an electric impedance microflow cytometry (EIMC) platform for the characterization of disease states of single cells. This platform entails a microfluidic device for a label-free and non-invasive cell-counting assay through electric impedance sensing. We identified a dimensionless offset parameter δ obtained as a linear combination of a normalized phase shift and a normalized magnitude shift in electric impedance to differentiate cells on the basis of their pathological states. This paper discusses a representative case study on red blood cells (RBCs) invaded by Plasmodium falciparum malaria parasites. Invasion of P. falciparum induces physical and biochemical changes on the host cells throughout a 48-h multi-stage life cycle within the RBC. As a consequence, it also induces progressive changes in electrical properties of the host cells .We demonstrate that the EIMC system in combination with data analysis involving the new offset parameter allows differentiation of Pf–invaded RBCs from uninfected RBCs as well as among different P. falciparum intraerythrocytic asexual stages including the ring stage. The representative results provided here also point to the potential of the proposed experimental and analysis platform as a valuable tool for non-invasive diagnostics of a wide variety of disease states and for cell separation.

It is important to generate fast fluid flow yet maintain low temperature rise for ac electrothermal ͑ac ET͒ pumping in microsystems with conductive fluids. This has been generally the limitation of ac ET driven micropump applications. We present an enhanced ac ET pumping mechanism using low voltage ac signals that can result in a small amount of temperature rise. Different from the published traveling wave and asymmetric electrode structures positioned on insulated flat surfaces, channels with a microgrooved surface are utilized in this study. The effects of the microgroove existence on the modification of the ET body force and recession of the vortex backflows are demonstrated. Forward and backward pumping modes are identified and analyzed. This mechanism utilizes a thin film of asymmetric electrode structure on the microgrooved channel floor that can be fabricated with common planar lithography technologies. This study demonstrates that using the microgrooved structure can increase pumping capacity by five to sixfold as compared to a planar electrode arrangement with the same effective dimensions.

Sickle-cell anaemia (SCA) is an inherited blood disorder exhibiting heterogeneous cell morphology and abnormal rheology, especially under hypoxic conditions. By using a multiscale red blood cell (RBC) model with parameters derived from patient-specific data, we present a mesoscopic computational study of the haemodynamic and rheological characteristics of blood from SCA patients with hydroxyurea (HU) treatment (on-HU) and those without HU treatment (off-HU). We determine the shear viscosity of blood in health as well as in different states of disease. Our results suggest that treatment with HU improves or worsens the rheological characteristics of blood in SCA depending on the degree of hypoxia. However, on-HU groups always have higher levels of haematocrit-to-viscosity ratio (HVR) than off-HU groups, indicating that HU can indeed improve the oxygen transport potential of blood. Our patient-specific computational simulations suggest that the HVR level, rather than the shear viscosity of sickle RBC suspensions, may be a more reliable indicator in assessing the response to HU treatment.

The human placenta plays a key role in reproduction and serves as a major interface for maternofetal exchange of nutrients. Study of human placenta pathology presents a great experimental challenge because it is not easily accessible. In this paper, a 3D placenta-on-a-chip model is developed by bioengineering techniques to simulate the placental interface between maternal and fetal blood in vitro. In this model, trophoblasts cells and human umbilical vein endothelial cells are cultured on the opposite sides of a porous polycarbonate membrane, which is sandwiched between two microfluidic channels. Glucose diffusion across this barrier is analyzed under shear flow conditions. Meanwhile, a numerical model of the 3D placenta-on-a-chip model is developed. Numerical results of concentration distributions and the convection–diffusion mass transport is compared to the results obtained from the experiments for validation. Finally, effects of flow rate and membrane porosity on glucose diffusion across the placental barrier are studied using the validated numerical model. The placental model developed here provides a potentially helpful tool to study a variety of other processes at the maternal–fetal interface, for example, effects of drugs or infections like malaria on transport of various substances across the placental barrier.

An optimization methodology is developed and applied to an ac electrothermal pump design with patterned microgrooved features. The microgrooved configuration can overcome the restrictions of the conventional planar configuration on pumping performance by diminishing fast backward flows and suppressing prolonged streamlines. At all frequency excitations (0.2–1000 MHz) and ion concentration conditions (5×10−3–0.1 M), the optimum microgrooved configuration generates much faster flow rate than planar configuration. This happens without additional increases in the maximum temperature values. The effects of elevated temperature on ac ET flow behavior is investigated and analyzed.