World Journal of Science and Technology 2012, 2(5):05-09 ISSN: 2231 – 2587 Available Online: www.worldjournalofscience.com

A review of recent advances in separation and detection of whole blood components

Doddabasavana Goud B., K. Padma Priya, and Nagabhushana Katte Department of ECE, JNTU College of Engineering, Anantapur – 515 002, India Department of ECE, Ballari Institute of Technology and Management, Bellary-583 104, Karnataka, India

Abstract Paper reviews the recent advances in separation and detection of whole blood components. It is intended for scientists in the field of separation science in biology, chemistry and micro-systems engineering. Recent advances/techniques of optical, magnetic, fluidic, electrical, ultrasonic, centrifugation, dielectrophoretic virtual pillar array, filtration, vibration and minor separation methods have been thoroughly discussed. Examples from the growing literature are explained with insights on separation efficiency and micro-engineering challenges. Latest separation/detection techniques and their applications have been discussed. Keywords: Whole blood, particle separation, detection.

INTRODUCTION

Blood is a valuable source to get various information about

our whole body. Though the elements of blood are usually keep within the constant range, the changes take place when abnormalities are found in our bodies. Therefore, when blood is examined, not only the sickness of blood but also various information on our bodies is obtained. By the blood examination, it is possible to detect a lot of diseases such as the lifestyle diseases and treat it early stage, and many people receive the inspection for sick prevention and a healthy check in recent years. However, it takes about one week by the results are obtained when inspecting in a small medical agency. The separation modules, selectively sort different kinds of particles, and this separation lead to immediate detection of the components for further analysis. Measuring instrument industry of medical, there are high expectations of developing the device that can be measured at once in place of diagnosis and treatment blood test. Hence it is very essential to know the various separation techniques, their advantages and disadvantages. This paper gives a complete review on different techniques used for separation/detection of whole blood components. This article also describes radical new ways to separate particles, improvement of older methods and novel, cost-effective manufacturing methods. Over the last ten years, point-of-use microreactors or point-of-care diagnostic tools have helped to reduce the need for intensive macrochemical plants or long diagnostic procedures. These so-called 'labs on a chip' are built out of several different modules, each

of them often achieving similar process functions as a macrochemical plant or a laboratory. Among these modules, the 'separation modules', selectively sort different kinds of particles, often immediately after synthesis or before analysis processes. Applications of separation techniques at the microscale are broad and versatile. Particle separation is a necessary preparation step in most biological microassays and common in microchemical processing. Microseparation techniques are also needed for the detection of cancer cells or the accumulation and counting of various types of cells and bacteria. For agrochemical, cosmetic and pharmaceutical companies, these techniques permit, after a chemical reaction, the separation, at production level, of solid products for post-treatment. In the food industry, potentially harmful bacterial activity is carefully monitored. Separation and enrichment of bacteria is necessary preliminary to analysis [2]. Monitoring of biological weapons is an important activity in the defence sector. In this field, separation is required to detect threatening agents such as Anthrax [3]. All these examples account for the tremendous need of portable, low-cost separation microdevices in a wide range of fields. Different separation methods in continuous and non-continuous are highlighted and illustrated by relevant examples: (1) Optical separation, (2) Magnetic separation, (3) Electrical separation (4)Fluidic separation (5)Dielectrophoretic virtual pillar array separation (6)Other type separation(7)Centrifugation separation (8)Filtering type separation (9)Vibration type separation. Past and Present Techniques This section describes different kinds of separation techniques within micro-channels currently developed as prototypes or already commercially available. A chart summarizing the techniques presented in this review is shown in Fig. 1.

Received: March 12, 2012; Revised: April 22, 2012; Accepted: May 20, 2012.

*Corresponding Author Doddabasavana Goud B Department of ECE, JNTU College of Engineering, Anantapur – 515 002, AP, India Email: dbg_rymec@rediffmail.com

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Fig 1. Classification of separation techniques

Continuous separation Optical separation This method uses optical tweezers as a manipulation tool by Ashkim, where tightly focused single laser beam is used to trap a single particle in micro fluidic system[4]. Advancement in optical manipulation allowed the creation of 3-D arrays of light traps (optical lattices) using holographic optical tweezers, phase contrast or diffractive optical element and multi-beam interferences. Optical fractionation systems are able to separate only one species from a mixture. In, however, separation of 4 different colloidal species is demonstrated using optical lattices created with an Acousto-Optic Deflector (AOD). AOD is used to control laser light and produce a complex potential energy landscape. AOD, a mixture of particles with 4 diameters (2.3, 3.0, 5.17 and 6.84 µm), pumped through micro channel is first focused in a single particle stream by an optical funnel. The smaller particles, which experience the weaker optical trapping force, leave the ramp first. In this way parallel separation of 4 subpopulations is achieved. This method shows throughput of 40 particles per second. The above throughput does not reach the throughput of Conventional Fluorescence-Activated Cell Sorters (FACS), it has higher throughput and where particles are optically interrogated one by one and directed into different outlets.

Fig 2. Optical fractionation concepts

3D optical lattice is introduced in the shared part of the chambers A, B, C and D allowing the separation of species according to their size or optical properties 3D optical lattice is reconfigurable which allows an easy updating of the selection criteria Optical separation separates only one component, system is complex, costlier and efficiency is up to 95% .

Magnetic separation

In conventional magnetic separation device a magnet is placed in the vicinity of a column containing the cells to be separated. Magnetically labeled cells are retained in the column; where as non-labeled cells will be flushed with the buffer allowing the immunological separation of species. The column is removed from the magnet in a second step and flushed to allow the collection of the sorted particles. This kind of separation is termed Magnetic Activated Cell Sorting (MACS). MACS is a batch process and might slow down analysis and limit the collection. A new attempt has made to fabricate continuous flow magnetic separation devices. A technique called ‘On-Chip free-flow magnetophoresis’ was demonstrated by Pamme et al[5,6]. In this mixture of different magnetic and non-magnetic particles is along the wall of a micro channel. A micro magnet placed upon the channel provides a non-homogeneous magnetic field gradient traverse to the laminar flow particles are deflected more or less from their path. The addition of spacers allows the collection of particles in separated outlet as shown in below figure 3.

Mixture of different magnetic and non-magnetic particles is injected in a micro-channel Depending on their size and magnetic properties the particles will be more or less deflected from their natural path due to the magnetic field, Non-magnetic particles will not be deflected. Addition of spacers permits the independent recollection of different species. Another type of continuous magnetic separation was demonstrated by Inglis et al[7]. In this example, some ferromagnetic strips fabricated in a micro channel provide an array like magnetic field pattern at a given angle to flow direction. Cells selectively tagged with magnetic nanoparticles deflect from the flow path to follow the strips. This method demonstrates the separation of WBCs as shown in the figure 4.

Illustration of a magnetophoretic separator Ferromagnetic wire is incorporated along the length of the microchannel. In the diamagnetic capture mode, an external magnetic field is applied normal to the x-axis of the microchannel In this case the red blood cells will be deflected from the ferromagnetic wire and will flow through the outlets 1 and 3 In the paramagnetic mode, the external magnetic field is applied normal to the y-direction of the

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microchannel, which forces the white blood cells to be deflected at this time from the ferromagnetic wire Reproduced with permission from reference [8] a) Diamagnetic capture mode b) Paramagnetic capture mode. In magnetic separation, process is slow i.e it consumes more time to separate, system is more complex ,costlier and efficiency is around 95%.

Electrical separation There are 2 main types of electric field-based manipulation, depending on the properties of the particles to be sorted. Electrophoresis, the movement of charged particles in a uniform electric field, is a very well known technique to separate different kinds of charged particles. Dielectrophoresis, the translation effect in a non-uniform electric field. Dielectrophoresis is to separate cells according to their size or dielectric properties. These techniques sometimes referred as stop-flow technique, relate to binary separation where a mixture is split into two subpopulations are usually returned in the channel. Sometimes particle separation is impossible. To overcome the above limitation, Yong et al [2], coupled antibody recognition and a Dielectrophoresis stop flow technique. Antibodies are coated above DEP arrays, isolated by a thin layer of SiO2 . The mixture of bacterial cells is injected into the channel when the DEP is actuated; all the cells are concentrated above the electrodes against the flow. During this time, the targeted cells bind themselves with the antibodies. When DEP is deactivated, the unbound cells flow away, leaving the targeted cells separated in the channel. These techniques report high efficiencies. First cells are trapped with positive dielectrophoresis(pDEP) Secondly only one subpopulation can be extracted from a heterogeneous mixture. Finally, voltages often have to be turned on and off to collect separated subpopulation, which can hamper the continuality of the process. Hydrodynamic forces have been coupled to dielectrophoresis to produce continuous particle separation. This technique uses electrodes to levitate particles to different heights depending on their dielectric properties parabolic flow allows the different particles to be dragged away at different velocities. This technique uses negative dielectophoresis to levitate particles above the electrodes and thus protects vulnerable biological particles from high electric fields. It has limitation in separation performance. Another kind of electrode arrangements have been successfully tested. Choi and park[9] proposed a trapezoidal planar electrode

array providing a specific electric field geometry in a micro channel. Li and Kaler[10] reported an ingenious ‘isomotive’ electrode arrangement for continuous flow separation. The use of dielectrophoretic barriers also been widely and successfully demonstrated. A dielectric barrier refers to electrodes mounted at the top and bottom in a micro-channel. This configuration deflects particles from the direction of flow. Other teams report the successful implementation of dielectrophoretic sorters, using improved DEP barriers[11-12] which double the maximum flow speed but it shows more temperature rise. A high speed cell-dipping system is proposed as shown in figure 6.

A Schematic diagram illustrating the concept of cell dipping Electrodes are mounted at the top and bottom of the microchannel. Population of cells is introduced in one of the inlets Electrodes, once activated, divert cells from their natural path Cells are guided to the reagent and from the reagent back to the buffer b Photograph of the experiment Transit time in the reagent is 0.3 s [14] Reproduced by permission of The Royal Society of Chemistry In electrical separation sometimes particle separation is impossible, reagent may change properties of whole blood, very strong electric field may damage the properties of blood components and some time it takes much time to separate. Fluidic separation Yamada and Seki[15] proposed a pinched flow separation device. The concept is shown in below fig.7. A mixture of different sized particles is injected into a buffer with the help of other fluid, particles are aligned to a sidewall of a pinched segment, which is subsequently broadened, at this point, hydrodynamic forces act differently on particles deflecting the small ones away from the big ones. Flow rates of the two inlets and the angle between the two segments are determining factors to sort the different size of particles. In 2005, Yamada proposed a 2 step technique called ‘Hydrodynamic Filtration’ to get rid of a second buffer. Another technique called deterministic lateral displacement tested by Huang[13]. Microposts are placed in rows within a micro channel. Each row of posts is shifted from the other by a distance which sets the separating size. The asymmetric bifurcation of laminar flow is to choose their path deterministically based on size. Liquid – liquid extraction is an another fluidic separation technique it is widely used in the chemical and biological industries and its efficiency of 97%. Schematic diagram illustrating the principle of pinched-flow fractionation, a) In the pinched segment, particles are aligned to one sidewall regardless of their sizes by controlling the flow rates from two inlets. b) Particles are separated according to their sizes by the

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spreading flow profile at the boundary of the pinched and the broadened segments

Dielectrophoretic Virtual Pillar Array separation The separation of microparticles, such as cells and functionalized beads is important to biochemical and medical applications. Microparticles have been separated by their own properties such as density, size, surface property, magnetism, dielectrophoretic (DEP) characteristics, and etc. Among them, separation by size difference has an important advantage which doesn't require additional labeling because many biological samples and functionalized beads have different sizes according to their own species. However, recent size-dependent particle separation using microstructures such as micropillar arrays [16] has a clogging problem in microstructures, which can cause malfunction of devices. The virtual pillar array induced by negative dielectrophoretic forces is shown in figure 8.

In order to reduce clogging in micropillar arrays, we have

proposed a virtual pillar array induced by negative dielectrophoretic (nDEP) forces and verified the capability of size-dependent particle separation in it with polystyrene beads. In this paper, we demonstrate continuous blood cell separation from diluted whole blood using the nDEP virtual pillar array.

The fabrication process can be classified into three steps: fabrication of the electrode layer, fabrication of the channel layer and bonding. Figure 9 Working principle of blood cell separation. In order to verify the feasibility of blood cell separation including RBCs and WBCs, we characterize separation of 5.7±0.28µm-diameter (5.7µm) and 8.0 ±0.80 µm-diameter(8.0 µm) Non-continuous separation Centrifugation This method uses the centrifugal force for the separation of mixture, and the rotation speed extend to several thousands rpm. The high-density particles in the mixture sink to bottom of centrifugal tubes by the force, and the solvent move to upper side. The advantage of this method is that it can separate a large amount of blood at one time. Filtering separation This method uses the porous membrane or nonwoven material, the particles are trapped by these filters. This filter is used for high-speed and it is not suitable for massive separation[17]. Most of the blood becomes dead volume after filter pores are clogged by blood cells in the filtration to avoid these problem to use serially connected membrane filters to increase collecting efficiency of separated plasma as shown in figure 10. The device has an upper and lower chamber for separating plasma from whole blood and device as pressure sensors of upper and lower chamber to avoid clogging. Clogging cells of filters and dead volume increased, when negative pressure at lower chamber (Pl) was large. On the other hand, most of whole blood at upper sections was transported to next filter and volume of extracted plasma was decreased, when negative pressure at upper chamber (Pu) was large. This same technique can adopt it for separating different microparticles in whole blood.

Vibration type particle separation In this separation particles are collected at the places that have maximum transverse velocity. At inside of a capillary, the particles are given the force towards antinodes by the inertial motion. Figure 11 shows the schematic diagram of vibration type particle separation principle. In this figure, the force given to the particle toward the capillary frame (arrow 1) will contain the two components of direction 2 and 3. The capillary do not have effective space for movement of direction 1, however particle has the little resistance to move in direction 3, which will give a net motion through direction 4 after a complete up and down cycle[18]. Figure 12 shows the schematic diagram of experimental system. The piezo-ceramic vibrator is driven by the waveform boosted by a power amplifier, and the waveform was generated by the function generator. At first, the applied frequency and applied waveform were selected by measurement of separation ability and separation time. After that, the experiment about effects by the

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measurement conditions were carried out and the conditions were distance between fixed points, sample viscosity and applied voltage. The acoustic radiation inside a capillary are usually performed by aligning a transducer into a micro-channel from one end to excite longitudinal modes of acoustic field through the tube. Another method is to use concave transducers to generate acoustic field to trap particles at the focus limiting the concentration area.

CONCLUSIONS A review of recent advances in microparticle separation and detection in whole blood components have been presented in this paper. The extensive literature present in the field as well as the wide range of applications, illustrates the tremendous interest drawn by microparticle separation. Separation is an important activity in the biological, medical and defence fields, to name but a few. Microparticle separation illustrates well the joined effort of different scientific communities that characterises the route towards integrated lab-on-a- chip devices. A photometric system is employed to detect the separated blood components of the column. REFERENCES [1] M. Kersaudy-Kerhoas, R.Dhariwal, M.P.Y.Desmulliez et

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