CDSship

Recent reports have shown increasing indication that the red blood cell (RBC) deformability depends on the health status of the patient [1, 2]. For example, diabetic [1] and malaria infected cells [2], have indices of deformability different of those of healthy cells. Investigation on RBC deformability requires two steps, separation of RBCs from the suspending plasma followed by deformation of RBC. Currently, this process is performed in two separate microdevice modules. Moreover, the blood samples used for this purpose need to undergo a number of preceding handling procedures (i.e. use of anticoagulants, plasma removal and centrifugation, etc). The methods traditionally used in these procedures may alter the original state of the cells of interest and consequently influence the results of any subsequent analysis. Therefore, new separation approaches able to be integrated with existing cell analysis strategies are essential to perform unaltered blood cell analysis.

The present project aims to design and implement a biochip able to perform in one single step both separation and deformation of cells within the plasma layer. This integrated new device will eliminate hours of lab time and reduce possible errors of procedure. As such, it will greatly increase the accuracy to diagnose a variety of diseases, including diabetes and malaria.

During the last years, members of this research project have been studying blood rheology in glass capillaries by means of a confocal micro-PIV system [3, 4, 10]. Very recently, due to the unique ability and benefits of elastomeric microchannels to efficiently control and replicate in vivo microvascular environments, we are now moving our research to study blood rheology in PDMS-based biomicrofluidics devices [11, 12]. Recent advances in microfluidics and microfabrication made possible to study both RBC deformation [2] and separation of RBCs from the suspending plasma [5, 11, 12].
Our recent studies [6, 11, 12] have shown evidence that it is possible to create an artificial plasma layer under appropriate hemodynamic and geometrical conditions. It has also been shown that the thickness of the plasma layer was enhanced downstream of an artificial stenosis (see Fig. 1). From this point of view, we believe that it is possible to take advantage of this hemodynamic finding to design a simple and highly reliable biochip capable of performing the steps of separation and deformation simultaneously.

The project starts with the design of cell separation module (CSM) prototypes based on our previous studies. The CSM will be manufactured in polydimethysiloxane (PDMS) by means of soft lithography techniques and will then tested using both physiological saline and healthy animal blood. Using the state of the art microvisualization systems, we will measure several hemodynamics parameters including, plasma layer thickness and plasma layer hematocrit. Based on the measured parameters we will optimize the design of the CSM and a cell deformation module (CDM) will be integrated into it. The CDM will consist of multiple arrays of straight microchannels to investigate the blood cells deformability. The integrated prototype will be optimized to evaluate at least a few hundreds of RBCs. The cell deformation index (CDI) will be evaluated using a micro-PTV system, composed of a high speed camera to capture images of RBCs, which will then be analysed by means of both manual and automatic image processing programs. Finally, the integrated chip for cell separation and deformation (CSD) will be tested clinically by performing experiments with healthy, diseased and drug-treated human blood cells.

Fig. 1 - The effect of a constriction on the plasma layer (Lima et al. 2009, unpublished data).

Goals:
The major goals and benefits of this project are:
• to understand important hemodynamic phenomena, such as the effect of geometrical and hemodynamic parameters responsible for the creation of the plasma layer, in order to optimize the performance of the biochip;
• to quantify the dynamical pressure-drop variation, velocity and deformability associated with the motion of single RBC passing through channel constrictions by using micro-PIV/PTV system;
• to evaluate CDI for healthy, diseased and drug-treated human blood cells without altering its original state, which is crucial for a more effective diagnosis;
• to, ultimately, develop a simple, affordable and portable biomedical tool for clinical haematology and pharmaceutical testing, which is able to perform in a single step both separation and deformation of cells within the plasma layer.


References:
1. Tsukada K., Sekizuka E., Oshio C and Minamitani H., 2001, Direct Measurement of Erythrocyte Deformability in Diabetes Mellitus with a Transparent Microchannel Capillary Model and High-Speed Video Camera System. Microvascular Research, Vol.61, 231-239.
2. Shelby J., White, J., Ganesan, K., Rathod, P., Chiu, T., 2003 A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes. PNAS 100: 14618-14622.
3. Lima, R., Wada, S., Tsubota, K.,Yamaguchi, T., 2006. Confocal micro-PIV measurements of three dimensional profiles of cell suspension flow in a square microchannel. Measurement Science and Technology 17, 797-808.
4. Lima, R., Wada, S., Takeda, M., Tsubota, K.,Yamaguchi, T., 2007. In vitro confocal micro-PIV measurements of blood flow in a square microchannel: the effect of the haematocrit on instantaneous velocity profiles. Journal of Biomechanics, 40: 2752-2757.
5. Faivre, M., et al., 2006. Geometrical focusing of cells in a microfluidic device: an approach to separate blood plasma. Biorheology 43 147-159.
6. Lima, R., Nakamura, M., Omori, T., et al., 2009. Microscale flow dynamics of red blood cells in microchannels: an experimental and numerical analysis. In: Tavares and Jorge (Eds), Advances in Computational Vision and Medical Image Processing: Methods and Applications, Springer, 203-220.
7. Oliveira, M. S. N., Rodd, L.E., McKinley, G. H., Alves, M. A., 2008, Simulations of extensional flow in microrheometric devices, Microfluidics and Nanofluidics, 5, 809.
8. Rodrigues, P., Fereira, M., Monteiro, J., 2009 Segmentation and Classification of Leukocytes Using Neural Networks: A Generalization Direction, In: Studies in Computational Intelligence, Springer, 373–392.
9. Pereira, A.; Fernandes, E., 2008 A reduction method for semi-infinite programming by means of a global stochastic approach. Optimization, 1–14
10. Lima, R., Ishikawa, T., Imai, Y., et al., 2008. Radial dispersion of red blood cells in blood flowing through glass capillaries: role of heamatocrit and geometry, Journal of Biomechanics, 41, 2188-2197..
11. Fujiwara, H., Ishikawa, T., Lima R., et al., 2009. Red blood cell motions in high-hematocrit blood flowing through a stenosed microchannel. Journal of Biomechanics, 42, 838-843.
12. Lima, R., Wada, S., Tanaka, S., Takeda, et al., 2008 In vitro blood flow in a rectangular PDMS microchannel: experimental observations using a confocal micro-PIV system. Biomedical Microdevices, 10(2), 153-167.