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Assessment Of Mems Blood Separation Techniques
 

Assessment Of Mems Blood Separation Techniques

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    Assessment Of Mems Blood Separation Techniques Assessment Of Mems Blood Separation Techniques Presentation Transcript

    • Blood Separation & Analysis Table of Contents 11/24/2008
        • Point of Care (POC) Testing & Products.
        • Types of Blood Analysis
        • Composition & Disorders
        • Conventional Blood Sampling & Separation
        • Developments in Cell Separation Techniques
      • - Based on size and form.
      • - Based on cell affinity.
      • ■ Summary of Methods
        • Challenges in cells separation & detection.
      Team Members: ○ Brandy Pearson ○ Joseph Attia ○ Zhiyu Chen ○ Abe Zandi Brandy/Zhiyu/Joseph/AZ
    • Point of care (POC) testing 11/24/2008 ■ Does not require permanent, dedicated space. ■ Eliminates need for nursing administrative efforts. ■ Focus on direct needs of patients. ■ Performed in proximity to the patient. ■ Provides “Fast Facts” to quickly identify and contain a disease. ■ Transfer from Lab/ field testing environment. ■ Reduce Cost of Diagnostic and testing ■ Efficient Data Management & Patients record keeping. ■ Flexibility of application (can be configured for other applications) ■ Plug-and-play interoperability for medical device communications (IEEE Std) ■ Weight, Size, Form, Battery Life, Reliability, Accuracy, Multiple input ■ Bio-Degradable/Disposable Sample or Applicator. AZ (Source: Building blocks for point-of-care boom, Ian Macfarlane & Fred Davis)
    • Successful POC Products
        • Blood glucose meters
          • Blood sample mixes with chemicals on a test strip, which produces an electric current, corresponding to the amount of glucose in blood. Provides results in ~5 seconds. Patient can monitor effects of dietary choices and medication.
        • Blood analyzers to measure cholesterol
          • Measures LDL (“bad cholesterol”), HDL (“good cholesterol”), Triglycerides, Ketones, and Glucose
        • Pregnancy test
          • A particular hormone called HCG reacts with monoclonal antibodies, creating a distinct color change
        • PH & Electrolyte Analyzer
          • Cycle time in ~70 seconds.
          • Uses Sealed Cartridge to Sample & Sense the chemicals
      11/24/2008 Brandy ( www.pointofcare.net/publications/index.htm )
    • 11/24/2008 Types of Blood Analysis
      • Blood Analysis – "blood work," is the most common laboratory test performed by physicians. There are several elements that we can examine:
        • Hematology – this looks at the cells within the blood, specifically red cells, white cells, and platelets. Hematologic studies can give great insight into a patient’s health, demonstrating conditions such as anemia, infection, and clotting disorders to name but a few.
      • B. Blood chemistry – this measures non-cellular components of the blood, including glucose, lipids, electrolytes, and certain hormones. Diabetes and thyroid disease are two common disorders that are detected with blood chemistry studies.
      • C. Serology – this series of tests specifically detect antigens and antibodies for certain disorders that are carried in a patient’s blood. Syphilis, HIV infection, and lupus are just some of the disorders that can be diagnosed with serologic studies.
      Only Hematology is considered for POC Testing AZ (Wheater’s Functional Histology, a text and colour )
    • 11/24/2008 Blood Composition & Diversity Cell population and subpopulation in normal blood 7- 20μm 6.5 to 8 4μm 1- 4μm 12- 15μm 10- 15μm 10- 15μm 10- 15μm AZ . Cell diameters too close to each other. POC resolution must be better less than 1 um (Blood-On-A-Chip, “Annual Rev. Mehmet Toner & Daniel Irima Biomed. Eng. 2005 7:77-103 ) Cell population diversity adds to the difficulty of separation
    • 11/24/2008 Blood Composition & Detection AZ RBC: Carry O2 WBC: Do not carry O2 (Wheater’s Functional Histology) Blood cells are different by Size, Composition, Color, Weight, Function, and percentage in sample RBC WBCs NEUTROPHILIC EOSINOPHILIC BASOPHILIC LYMPHOCYTE MONOCYTE MONOCYTE (Davidson , 2007) Cell Detection is expensive and Lab-oriented (Wheater’s Functional Histology)
    • Conventional Blood (RBC/WBC) Separation 11/24/2008
        • Membrane Separation
        • Centrifugal Separation
      (www.AlfaLaval.com) (Diehl , et al.) Zhiyu/Joseph
    • 11/24/2008 11/24/2008 Blood Sample Preparation (Centrifugal) (*)
        • ■ Collect Human Blood
          • Add BD Vacutainer® tube containing Acid-citrate-dextrose (ACD) solution A as an anticoagulant.
        • ■ Centrifuge I (rough separation)
          • Spin down at 1800rpm for ~15min
          • After centrifugation, one can distinguish:
          • - Plasma (upper layer of clear fluid)
          • - Buffy coat (a thin layer making up less
          • than 1% of the total volume of the blood sample,
          • with most of the white blood cells and platelets)
          • - Red Blood Cells (lower layer of red fluid)
          • ( UCLA Immunogenetic Lab)
      Zhiyu http://www.phlebotomypages.com/blood_panel.htm
    • 11/24/2008 11/24/2008
        • ■ Collect Buffy Coat
          • Using pipette to collect the entire layer of buffy coat. Add Phosphate buffer saline (PBS) to dilute. Mix by pipetting up and down.
        • ■ Centrifuge II ( Separate PBMC)
          • Pipette Buffy coat + PBS slowly to a tube with Ficoll-Paque to avoid mixing
          • Ficoll-Paque is a solution used in centrifuge to separate Peripheral Blood Mononuclear Cells (PBMC), which include lymphocytes and monocytes.
          • - Spin down at 1500rpm for ~20min
          • After centrifugation, one can distinguish:
          • - Medium/Serum (upper layer of clear fluid)
          • - PBMC (a thin milky-white layer between Serum and Ficoll)
          • - Ficoll (middle layer of clear fluid)
          • - Red Blood Cells (lower layer of red fluid)
          • ( UCLA Immunogenetic Lab)
      Blood Sample Preparation (Centrifugal) (*)
          • (Zhiyu, UCLA Immunogenetic Lab)
      Zhiyu
    • 11/24/2008 11/24/2008
        • ■ Collect Middle Layer containing PBMC
          • Using pipette to collect the middle layer. Add Phosphate buffer saline (PBS) to dilute.
        • ■ Centrifuge III (Purify PBMC)
          • -Spin down at 1500rpm for ~10min
          • After centrifugation, one can distinguish:
          • - Upper layer (mainly PBS)
          • - White Blood Cells in the bottom
        • ■ Wash the WBCs
        • - Remove upper layer
        • - Add PBS to re-suspend the white blood cells
        • - Repeat Centrifuge to increase sample purity .
        • (1500rpm for 5min )
          • ( UCLA Immunogenetic Lab)
      Blood Sample Preparation (Centrifugal) (*)
          • (Zhiyu, UCLA Immunogenetic Lab)
      Zhiyu
    • 11/24/2008 11/24/2008
        • ■ Collect PBMC
          • Remove the upper layer (PBS) after centrifuge;
          • Dilute with Roswell Park Memorial Institute medium (RPMI) and human AB serum (HAB) solution (9:1);
          • Re-suspend the cells;
        • ■ Cell culture to separate lymphocytes and monocytes
          • Store the PBMC with PRMI/HAB at 37 ℃ for several hours. After that:
          • - Monocytes will adhere to the inner surface.
          • - Lymphocytes will be in the solution.
        • ■ Collect Solution with Lymphocytes
      Blood Sample Preparation (Centrifugal) (*)
        • Notice that conventional method is:
        • - Expensive
        • - Lab-oriented
        • -Time consuming
          • ( UCLA Immunogenetic Lab)
          • (Zhiyu, UCLA Immunogenetic Lab)
      Zhiyu
    • 11/24/2008 Multi-Functionality of POC AZ ( “ Understanding Complete Blood Count “ (CBC) Report; Sonora Quest Laboratories, Arizona, CA.) Many Disorders One Platform GOAL: Maximize associated disorders through Similar Processes of separation and detection
    • Cell Separation Techniques Research & Development 11/24/2008 ■ Cell separation techniques can be broadly classified into two categories: B. Sized-Based Methods: - Relatively fast and simple. - Type of cells is determined and separated according to their cell size, shape and other physical properties - Disadvantage of this method is its low specificity for cell separation, - Separation is not too sensitive to small size variation of cells. A. Affinity-Based Methods: - Particle separation due to affinity of antigen to antibodies in the sample with high specificity and selectivity - The isolated cells may suffer from damages. - High cost and complicated processes such as immunoreactions and elution (extraction by means of solvents) of cells from the capturing antibodies create challenges (Zheng, Siyang, Raylene Yung, et al. 2005) AZ (Zheng, Siyang, Raylene Yung, et al. 2005)
    • Cell Separation Techniques Research & Development 11/24/2008 ■ Cell separation techniques can be broadly classified into two categories: A. Techniques based on size, shape & density. B. Techniques based on cell affinity (chemical, electrical, or magnetic) Magnetophoresis Electrophoresis Adhesion-Based Florescence-Based Capillary Electrophoresis & Capillary cIEF Electro-hydrodynamic AZ Obstacle & Sieve (Zheng, Siyang, Raylene Yung, et al. 2005)
    • 11/24/2008 Size & Form-Based Separation Obstacle/Sieve
        • Deterministic Lateral Displacement
          • Utilized at right: Laminar flow is pushed past a collimated series of obstructions. When particles are smaller than the width of the streamlines formed, particles will zigzag between obstacles and return to its initial row. When particles are larger than the streamline width, they collide and are displaced, separating them from the rest of the flow. See Figure at bottom right. (Zheng 2005)
      Separation of leukocyte (highlighted in green) from erythrocyte (red) via lateral displacement. An overlay of multiple video frames. ( Zheng 2005)
        • Physical “Sieve”
          • Utilized at right: 4 successively narrow arrays of parallel channels from 20um to 2.5 um. Acts as a sieve, trapping larger cells upstream. Requires cells to be significantly different in size. (Mohamed 2004 )
      Lateral Displacement: Flow is confined to one of three streamlines denoted as 1, 2 and 3. (Huang LR, Cox ED)
        • Potential Problems:
          • Stiction Effectively Reduced with Dilution
            • Stiction occurred at entrance of blood flow into device.
            • The device made of hydrophobic Teflon and cells are hydrophilic, which should yield minimal cell stiction.
            • Minimal cell stiction with a 2X dilution factor---Still requires dilution.
            • Could a super hydrophobic material be used?
            • (Zheng 2005)
      Brandy
    • Size & Form-Based Separation Electro hydrodynamic Flow
        • Blood enters cylindrical chamber where a needle tip is placed at an angle near the fluid surface
        • Voltage applied to the needle causes the fluid to rotate
        • Opposing centrifugal forces at the bottom of the cylinder separates particles from fluid (i.e. WBC, RBC and plasma)
        • Requires High electric Field (kV/m)
      • (Arifin, 2007)
      11/24/2008 Successive images obtained by high speed video microscopy at 60 frames/ s showing the spiral-like trajectory of the RBCs near the bottom of the microfluidic chamber toward a stagnation point. The applied field and frequency are 286 kV/m and 60 kHz, respectively, and the initial hematocrit is 0.4%. (Arifin, 2007) Joseph Einstein’s Tea Leaf Paradox Red blood cells would be pulled to the outside wall of the chamber owing to centrifugal force. But because of a phenomenon called the "tea leaf paradox," the particles are instead pulled inward near the bottom of the chamber. (Arifin, 2007) (Arifin, 2007)
    • 11/24/2008 Fluorescence-Based Separation
        • Staining blood cells with fluorescent dye, Acridine Orange (AO) and sensing them optically. AO binds strongly to RNA and single-stranded DNA, since mature erythrocytes lack DNA and RNA, only leukocytes are stained. Differentiation of leukocytes is done by analyzing the signal of red fluorescence.
        • Potential Problems:
          • Blood cell sedimentation on micro fluidic device
            • Eliminated via high throughput of ~100 leukocytes/second
          • Photo bleaching of cells due to laser light
            • Time constant for photo bleaching on order of seconds, while cells pass through detection zone on order of milliseconds. Photo bleaching has no time to occur.
          • Requires external optical system
            • Can it be miniaturized?
            • (Zheng 2007)
      Above: Cell count corresponding to signal intensity from photodiode detector with red emission filter. Peaks correspond to different types of leukocytes. (Zheng 2007) Dominated by lymphocytes. Dominated by monocytes and granulocytes. Above: Optical system used to sense and analyze leukocytes. (Zheng 2007) Brandy
    • 11/24/2008 Adhesion-Based Separation
        • Binding/Adhesion occurs by antibodies immobilized on device surface. (Similar to chromatography)
        • Able to separate leukocyte populations that have ~same size and density
        • No need to preprocess blood sample with florescent or magnetic tags.
      Micro fabricated system Adhesion based separation method: It mimics the leukocyte recruitment from blood vessel at sites of inflammation, where leukocyte slow down by transient attachment and attachment of antigen/antibody bonds between cell membrane and endothelium . By coating the antibody on channel wall with sufficient density, it is expected that target cells slow down to terminal velocity where they are separated ( 9 th International Conference) Micro fabricated Prototype cell separation column ( 9 th International Conference on Miniaturized system for chemistry & Life Sciences; Oct 9-13, 2005 USA) Joseph
    • 11/24/2008 Magnetophoresis Separation
        • Cell separation using magnetic beads coated
      • with antibodies specific for surface antigens
      • of cells of interest.
        • Cells bind to the beads and can be separated
      • by a magnetic field.
        • Magnetic sorting can be easily miniaturized.
        • Potential Problems:
          • -Efficiency separation in continuous system.
          • -Magnetic filtration system has been achieved
          • with up to ~95% efficiently.
          • (Inglis 2006)
      Separation of immunogenic T cells from whole blood by: (1) flowing coated paramagnetic beads with Protein-A/anit-human CD3 into device and immobilize by external magnet, (2) flow whole blood over the beads to capture the T cells, (3) remove external magnet and cells flushed out of channel. Only 40% efficiency. (Furdui 2004) At Left: Whole blood flows over surface of ferromagnetic strips. Cells are labeled with anti-CD45 conjugated super paramagnetic nanoparticles. Strips are positioned at an angle to deflect the labeled cells. Tagged leukocyte Combination fluid flow
          • (Inglis 2006)
      Brandy
    • Non-Intrusive Magnetophoresis Separation
        • Cell Separation purely based on diamagnetic/paramagnetic properties of WBC/RBC (s)
        • Applied magnetic field imposes force on blood cells and cells are diverted
        • Direct and continuous separation of cells
        • Difficulties
      • - Red blood cells must be deoxygenated in order for magnetic field to have any effect
      11/24/2008 Magnetophoretic microsystem: ( a ) microsystem with bias field structure, ( b ) cross section of microsystem showing magnetic elements beneath the microchannel and ( c ) magnified view of microfluidic channel showing the bias field, magnetic elements and forces on red and white blood cells (RBC and WBC). (Furlani 2007) Joseph (Furlani 2007)
    • 11/24/2008 Electrophoretic Separation
        • Particles with an induced electric polarization, such as dipoles, quadruples, and octopoles, can be trapped within a non-uniform electric field.
        • Negative DEP forces cause polarized objects
      • to be repelled away from an electrode, while positive DEP forces attract objects toward an electrode.
        • DEP depends on the existence of a gradient in an electric field.
        • “ AC” voltage is usually employed.
      • (Voldman 2002)
      An array of DEP traps, each comprising four trapezoidaly arranged electrodes of opposite polarity, was fabricated to create a non-uniform, quadruple electric field. (Voldman 2002) Two cells are loaded into the trap at low flow rate. The application of higher flow rate results in the ejection of one cell (dark grey arrow) from the trap leaving the other cell behind (grey arrow). (Voldman 2002) AZ (Voldman 2002)
    • 11/24/2008 Capillary electrophoresis (CE) AZ ■ Uses capillaries (e.g. porous fused-silica) to separate a complex array of large/small molecules ■ High electric field strengths are used to separate molecules based on differences in charge, size and hydrophobicity. ■ Apply pressure, vacuum or voltage to end of the capillary vial sample too separate. ■ Types of capillary and electrolytes determine the separation techniques: Surfactants are added to the buffer solution at concentrations that form micelles. Separation Principle: differential partition between the micelle and the solvent. Micelles: An aggregate of surfactant molecules dispersed in a liquid colloid. ○ Micellar Electrokinetic Capillary Chromatography (MECC ) Solutes partition with moving oil droplets in buffer. ○ Micro Emulsion Electrokinetic Chromatography (MEEKC) Include electro-osmosis, electrophoresis and chromatography (adsorptive materials separation) ○ Electro-kinetic Chromatography (EKC): Based on the migration of the sample components between leading and terminating electrolytes ○ Isotacho-phoresis (ITP) Electrophoresis in a pH gradient generated between the cathode and anode. A solute will migrate to a point where its net charge is zero. Sample focused into tight zone. ○ Capillary Isoelectric Focusing (CIEF) Uses polymers in solution to create a molecular sieve. ○ Capillary Gel Electrophoresis (CGE) Based on differences in the charge-to-mass ratio of the analytes ○ Capillary Zone Electrophoresis (CZE ( Beckman Coulter Corporation, “Capillary Electrophoresis: A Simple Technique”)
    • 11/24/2008 Capillary Isoelectric Focusing (cIEF) Separation
        • Relies on equilibrium between diffusion and electrophoresis of a species within pH and voltage gradients to accomplish separations.
        • Individual species are driven to a stationary location within the separation column that corresponds to the Isoelectric point (pI, the pH at which the net charge on the sample goes to zero) of the respective species. This equilibrium behavior results in a high density of tightly-focused sample species spatially distributed within the separation column.
        • A mixture of protein samples is subjected to a strong electric field in a medium designed to have a linear pH gradient. At the molecular level, samples Electrophoretically migrate along the channel until they reach the point in the pH gradient that is equal to their Isoelectric pH (pI), where the protein no longer has a net charge. At the pI, the Electrophoretic force on the protein due to the applied electric field is zero. Once the separation is complete and all samples have reached their respective Isoelectric points, the separation is simply a stationary spatial distribution.
      • ( Herr, 2007)
      An axial electric field has been applied, resulting in the formation of an axial pH gradient within a background field of ampholytes (represented by the white/black color gradient). Proteins initially distributed homogeneously in the column migrate to their respective pI location (represented by circles w-z). ( Herr, 2007) Capillary cartridge AZ ( Herr, 2007)
    • 11/24/2008 Summary of Methods ● PH dependent ● Low efficiency ● Requires Pressure or Vacuum. ► Laminar Flow ► Moderate throughput Capillary Electrophoresis : Capillary Isoelectric Focusing (cIEF) ● Packed bed design; Difficult to miniaturized ● Antibody tags : Requires lab preparation ● Cell populations have same size or density ► No need for incubation of staring cell ► Provides high purity (>95%) ► Provide high throughput (108-109 cells/hour) Surface Adhesion ► Electro-osmotic flow potential ► Laminar Flow Florescence Activated Cell Sorting (FACS) ► Post Size and Diameter ► Particle size differentiation ► Used mostly on protein & Peptides Electrophoresis ● Require Cell-Specific Marker. ● Requires exact cell position relative to the magnetic field, ● Low efficiency in general. ● Maximum two particle sorting ● AC Power ● Antibody tags : Requires lab preparation ● High Cost (Not feasible commercially) ● Difficult to miniaturize ● Non-Intrusive method: Blood must be deoxygenated. ► Particle size differentiation ► Magnetic properties ► High efficiency possible Magnetophoresis. - Intrusive - Non-Intrusive ● Require Cell-Specific Marker. ● Isolated cells suffer from damage. ● Low efficiency ● High Cost (Not feasible commercially) ► Laminar Flow ► Continuous (flow through) system Magnetic Activated Cell Sorting (MACS) ● Requires high electric Field ● Difficult to miniaturize ► High efficiency Electro-dynamic Flow ● Low specificity of cell separation. ● Insensitive to small differences in Size & Form ► Laminar Flow ► Cell-Specific Markers not required Size-Based WEAKNESS A Good POC Candidate? IMPORTANT PARAMETERS & FEATURES SEPARATION METHOD` AZ
    • Challenges in blood cells separation and detection 11/24/2008 ■ Challenge: Massive numbers & diversity of cell types complicate precise identification of the target cells from blood sample. Approach: Multiple sampling (within the same package) and using similar method to identify various cell properties. ■ Challenge: Many cells can only be identified by the presence of specific protein markers (Antigen/Antibody) on the surface of cells Approach: Use intrinsic cell properties (Mobility, polarity etc.) that eliminates need for specific markers. Integrate a combination of cell separation techniques in single platform. ■ Challenge: Some processes require longer time of sorting & detection (e.g. sample culture) Approach: New innovations are key to success in reducing time of processes ■ Challenge: Minimum handling to reduce contamination and exposure Approach: Self contained, discard able, bio-degradable test sample; Lab-on-Chip ■ Challenge: Test Package is inadequate for Field use ( Too big, AC power, handling, Non-portable) Approach: Reduce complexity of design with fresh scientific approach. DC Power. Portable platform ■ Challenge: High Cost($) & Low efficiency Approach: Simple Design & Instrumentation (Mfg. for COTS). Improved detection system ■ Challenge: High Reliability & Repeatability ( particle count > 50000 Cells/sec) Approach: Miniaturization of improved instruments. New techniques of detection. Increase precision in nanoscale domain (CNT, naowires,..etc) Joseph/AZ
    • References
        • Arifin, Dian R., Leslie Y. Yeo, and James R. Friend Micro/Nanophysics Research Laboratory, Department of Mechanical Engineering, Monash University, Clayton, VIC 3800, Australia
        • Beckman Coulter Corporation, “Capillary Electrophoresis: A Simple Technique” ”;http://www.beckman.com/resourcecenter/labresources/ce/cedefinitionmodes.asp. 11/2007
        • Blood-On-A-Chip; Annual Rev. Biomed Eng. 2005. 7:77-103; Memet Toner & Daniel Irima
        • Building Blocks for point-of-care boom, Ian Macfarlane & Fred Davis, IDT Technology; Jan/Feb 2002
        • Furdui VI, Harrison DJ 2004. Immunomagnetic T cell capture from blood for PCR analysis using microfluidic systems. Lab Chip , 4:614-18.
        • Herr, Amy E., Molho, Joshua I., et al. Mechanical Engineering Department, Stanford University Stanford, CA 94305-4021
        • Huang LR, Cox EC, Austin RH, et al 2004. Continuous particle separation through deterministic lateral displacement. Science , 304: 987-90
        • Inglis DW, Riehn R, Austin RH, et al. 2004. Continuous microfluidic immunomagnetic cell separation. Appl Phys Lett , 85:5093-5.
        • “ Introduction to Cell and Virus Structure”, Michael W. Davidson & National Magnetic Field Laboratory,
        • Magnetophoretic Blood Separation at the Microscale, E P Furlani J. Phys. D: Appl. Phys. 40 (2007) 1313–1319
        • Mohamed H, McCurdy LD, Szarowski DH, et al. 2004. Development of a rare cell fractionation device: Application for cancer detection. IEEE Trans Nanobioscience , 3:251-6.
        • “ Understanding Complete Blood Count (CBC) Report; Sonora Quest Laboratories, Arizona, CA
        • Voldman J, Gray ML, Toner M, et al. 2002. A micro fabrication-based dynamic array cytometer. Anal Chem , 74:3984–90. Reproduced with permission. Copyright © 2002 American Chemical Society.
        • Wheater’s Functional Histology, a text and color atlas, p 57. 5 th Edition, Barbara Yong & Alan Stevens
        • Zheng, Siyang, Jeffrey Lin, et al. 2007. Fluorescent Labeling, Sensing and Differentiation of Leukocytes from Undiluted Whole Blood Samples. The 14 th International Conference on Solid-State Sensors, Actuators and Microsystems, Lyon, France, June 10-14, 2007.
        • Zheng, Siyang, Raylene Yung, et al. 2005. Deterministic Lateral Displacement MEMS Device for Continuous Blood Cell Separation. MEMS 2005 Miami Technical Digest , 851-4.
      • Florida State University.; 2007
      11/24/2008 Brandy/Zhiyu/Joseph/AZ