2. 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)

5. 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

6. 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)

12. 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

13. 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)

14. 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)

22. 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”)

24. 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

25. 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