Sehyun Shin

New guidelines for hemorheological laboratory techniques

This document, supported by both the International Society for Clinical Hemorheology and the European Society for Clinical Hemorheology and Microcirculation, proposes new guidelines for hemorheolog ...

Separation of platelets from whole blood using standing surface acoustic waves in a microchannel

Platelet separation from blood is essential for biochemical analyses and clinical diagnosis. In this article, we propose a method to separate platelets from undiluted whole blood using standing surface acoustic waves (SSAWs) in a microfluidic device. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with interdigitated transducer (IDT) electrodes patterned on a piezoelectric substrate. To avoid shear-induced activation of platelets, the blood sample flow was hydrodynamically focused by introducing sheath flow from two side-inlets and pressure nodes were designed to locate at side walls. By means of flow cytometric analysis, the RBC clearance ratio from whole blood was found to be over 99% and the purity of platelets was close to 98%. Conclusively, the present technique using SSAWs can directly separate platelets from undiluted whole blood with higher purity than other methods.

Parameterization of red blood cell elongation index – shear stress curves obtained by ektacytometry

Abstract Measurement of red blood cell (RBC) deformability by ektacytometry yields a set of elongation indexes (EI) measured at various shear stresses (SS) presented as SS-EI curves, or tabulated data. These are useful for detailed analysis, but may not be appropriate when a simple comparison of a global parameter between groups is required. Based on the characteristic shape of SS-EI curves, two approaches have been proposed to calculate the maximal RBC elongation index (EImax) and the shear stress required for one-half of this maximal deformation (SS1/2): (i) linear Lineweaver-Burke (LB) model; (ii) Streekstra-Bronkhorst (SB) model. Both approaches have specific assumptions and thus may be subject to the measurement conditions. Using RBC treated with various concentrations of glutaraldehyde (GA) and data obtained by ektacytometry, the two approaches have been compared for nine different ranges of SS between 0.6–75 Pa. Our results indicate that: (i) the sensitivity of both models can be affected by the SS range and limits employed; (ii) over the entire range of SS-data, a non-linear curve fitting approach to the LB model gave more consistent results than a linear approach; (iii) the LB method is better for detecting SS1/2 differences between RBC treated with 0.001–0.005% glutaraldehyde (GA) and for a 40% mixture of rigid cells but is equally sensitive to SB for 10% rigid cells; and (iv) the LB and SB methods for EImax are equivalent for 0.001% and 0.003% GA and 40% rigid, with the SB better for 0.005% GA and the LB better for 10% rigid.

Density-dependent separation of encapsulated cells in a microfluidic channel by using a standing surface acoustic wave

This study presents a method for density-based separation of monodisperse encapsulated cells using a standing surface acoustic wave (SSAW) in a microchannel. Even though monodisperse polymer beads can be generated by the state-of-the-art technology in microfluidics, the quantity of encapsulated cells cannot be controlled precisely. In the present study, mono-disperse alginate beads in a laminar flow can be separated based on their density using acoustophoresis. A mixture of beads of equal sizes but dissimilar densities was hydrodynamically focused at the entrance and then actively driven toward the sidewalls by a SSAW. The lateral displacement of a bead is proportional to the density of the bead, i.e., the number of encapsulated cells in an alginate bead. Under optimized conditions, the recovery rate of a target bead group (large-cell-quantity alginate beads) reached up to 97% at a rate of 2300 beads per minute. A cell viability test also confirmed that the encapsulated cells were hardly damaged by the acoustic force. Moreover, cell-encapsulating beads that were cultured for 1 day were separated in a similar manner. In conclusion, this study demonstrated that a SSAW can successfully separate monodisperse particles by their density. With the present technique for separating cell-encapsulating beads, the current cell engineering technology can be significantly advanced.

Comparison of three commercially available ektacytometers with different shearing geometries

In December 2008, the International Society for Clinical Hemorheology organized a workshop to evaluate and compare three ektacytometer instruments for measuring deformability of red blood cells (RBC): LORCA (Laser-assisted Optical Rotational Cell Analyzer, RR Mechatronics, Hoorn, The Netherlands), Rheodyn SSD (Myrenne GmbH, Roetgen, Germany) and RheoScan-D (RheoMeditech, Seoul, Korea). Intra-assay reproducibility and biological variation were determined using normal RBC, and cells with reduced deformability (i.e., 0.001-0.02% glutaradehyde (GA), 48 degrees C heat treatment) were employed as either the only RBC present or as a sub-population. Standardized difference values were used as measure of the power to detect differences between normal and treated cells. Salient results include: (1) All instruments had intra-assay variations below 5% for shear stress (SS)>1 Pa but a sharp increase was found for Rheodyn SSD and RheoScan-D at lower SS; (2) Biological variation was similar and markedly increased for SS<3-5 Pa; (3) All instruments detected GA-treated RBC with maximal power at 1-3 Pa, the presence of 10% or 40% GA-modified cells, and the effects of heat treatment. It is concluded that the LORCA, Rheodyn SSD and RheoScan-D all have acceptable precision and power for detecting reduced RBC deformability due to GA treatment or heat treatment, and that the SS range selected for the measurement of deformability is an important determinant of an instruments power.

Magnetic separation of malaria-infected red blood cells in various developmental stages

Malaria is a serious disease that threatens the public health, especially in developing countries. Various methods have been developed to separate malaria-infected red blood cells (i-RBCs) from blood samples for clinical diagnosis and biological and epidemiological research. In this study, we propose a simple and label-free method for separating not only late-stage but also early-stage i-RBCs on the basis of their paramagnetic characteristics due to the malaria byproduct, hemozoin, by using a magnetic field gradient. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with a ferromagnetic wire fixed on a glass slide. To evaluate the performance of the microfluidic device containing the ferromagnetic wire, lateral displacement of NaNO2-treated RBCs, which also have paramagnetic characteristics, was observed at various flow rates. The results showed excellent agreement with theoretically predicted values. The same device was applied to separate i-RBCs. Late-stage i-RBCs (trophozoites and schizonts), which contain optically visible black dots, were separated with a recovery rate of approximately 98.3%. In addition, using an optimal flow rate, early-stage (ring-stage) i-RBCs, which had been difficult to separate because of their low paramagnetic characteristics, were successfully separated with a recovery rate of 73%. The present technique, using permanent magnets and ferromagnetic wire in a microchannel, can effectively separate i-RBCs in various developmental stages so that it could provide a potential tool for studying the invasion mechanism of the malarial parasite, as well as performing antimalarial drug assays.

Toxic effects of silver nanoparticles and nanowires on erythrocyte rheology

Rapid developments in the food applications of silver nanomaterials (Ag-NMs) have resulted in concerns related to the risk of overexposure of human blood. We investigated the effect of size and aspect ratio of Ag-NMs on rheological characteristics of human erythrocytes, including hemolysis, deformability, aggregation, and morphological changes. Red blood cells (RBCs) were exposed to two different sizes of spherical particles (d∼30 nm or 100 nm) or nanowires (d∼40 nm, l-2 μm in length) at a range of concentrations and incubation times. The concentrations of Ag-NMs were carefully chosen to avoid any hemorheological alteration due to hemolysis. Rheological properties were measured using microfluidic-laser diffractometry and aggregometry. RBC deformability apparently decreased after treatment with a low concentration of Ag-NPs for a short exposure time. However, RBC aggregation was significantly altered after treatment with a low concentration of either Ag-NWs or large Ag-NPs compared to small Ag-NPs. Additional experiments with Ag ions confirmed that the observed rheological changes were mainly caused by the Ag-NMs rather than the Ag ions. These hemorheological findings provide a better understanding of the interaction between RBCs and Ag-NMs and will help in assessing the risk of nanomaterial toxicity in blood.

Measurement of erythrocyte aggregation in a microchip stirring system by light transmission

In the analysis of red blood cell (RBC) aggregation using optical detection, various shearing methods have been used to disperse RBCs in confined geometries. This study investigated RBC aggregation measurement in a microchip-stirring system by analysis of light transmission. A stirring-aided disaggregation mechanism in a microchip, consisting of a flat-cylindrical test chamber (D=4 mm, H=0.3 mm) and a magnetic stirrer (d=0.14 mm, l=2.2 mm), was used to generate a given shear which was large enough to disperse RBC aggregates, but not large enough to cause any mechanical hemolysis of cells. After stirring for 10 s followed by an abrupt halt of the stirring, the intensity of the light transmitted through a microchip was measured with respect to time and analyzed. A comparative study was conducted with varying test chamber height and hematocrit. The AI and t1/2 as typical aggregation indices obtained by analysis of transmitted light, which showed a good reproducibility (coefficient of variation (CV)<2.8%, n=10), also were found to be nearly independent of the chamber dimensions (CV<3.4%). The present aggregometry also showed the similar results of aggregation indices with varying hematocrits compared to those obtained using a laser-assisted optical rotational cell analyzer (LORCA). The essential feature of the present design is the adoption of a disposable microchip requiring a minimum blood sample volume as small as 6 mul, which enables it to be used easily in a clinical setting.

Advances in the measurement of red blood cell deformability: A brief review

Red blood cells (RBCs) exhibit a unique deformability, which enables them to change shape reversibly in response to an external force. The deformability of RBCs allows them to flow in microvessels while transporting oxygen and carbon dioxide. In this review, we discussed the major determinants of RBC deformability, which include cell geometry, internal viscosity, rheological properties of the membrane, osmotic pressure, calcium, nitric oxide, temperature, ageing, and depletion of adenosine triphosphate. Additionally, we highlighted the various methods and techniques used to measure RBC deformability. Individual cell analyses (pipette aspiration and optical tweezers) and bulk cell analyses (ektacytometry, multiple channels) were described and compared. Finally, we reviewed the correlation between RBC deformability and clinical outcomes such as diabetic microangiopathy.

A transient, microfluidic approach to the investigation of erythrocyte aggregation: The threshold shear-stress for erythrocyte disaggregation

Detailed analysis of red blood cells (RBCs) aggregation is often required in various clinical studies. Most conventional aggregation indices are dimensionless values and not available for comparison of across studies. Quite recently, we have developed microfluidic aggregometry that enables us to yield a critical shear-stress that are required to aggregate RBCs under the shearing hydrodynamic force. The present study investigated the relationships between the values of the critical shear-stress and conventional aggregation indices by comparing the critical shear-stress measured by the microfluidic aggregometry with the threshold shear-stress measured using a LORCA aggregometer. The results showed that the critical shear-stress did not vary with the hematocrit value while the threshold shear-rate decreased with the hematocrit value. The threshold shear-stress also showed the same hematocrit-independence as the critical shear-stress. These findings assist in rheologically validating the critical shear-stress, as defined in the microfluidic aggregometry, within the present range of hematocrit values.