Multi-channel Cell Counter Utilizing The Aperture Impedance Technique

1. Multi-channel Cell Counter Utilizing The Aperture Impedance Technique Aaron Lee & Dr. Ash M. Parameswaran Simon Fraser University School of Engineering Sciences Burnaby, B.C. Canada V5A 1S6 Email: cleek@sfu.ca This work is sponsored by Brain Insights, California

2. Overview • Introduction • Centralized approach • Clinical facts • Techniques of cell counting • Electrical and physical relationships • Disposable unit design • Conclusion

3. Thesis Concentration Construction, modeling and testing of the disposable unit and the electronics

4. Introduction • Most people have blood test at some point in their lives • Blood is the vital fluid of our body and the quality of blood is an indication of health • Measured in number of cells per cubic millimeter of blood

5. Centralized approach • Most blood cell counting today is done by sending the blood samples to a centralized laboratory • Very complex system and required skilled personnel to operate • Long turn-around time • Patient has to visit another time

6. 18 cell sizes result and histograms Dimensions: 37x47x38(cm) Weight: 18 kg net Power: AC No portable blood counter in the market Commercial blood cell counter

7. Our challenges • Shortens the turn around time • Reduce the cost so clinics can afford to own the blood cell counter • Miniaturize the testing equipment • Maintain or improve accuracy

8. Blood cell sizes and their normal ranges

9. Diseases of the Blood

10. Cell count techniques • Electrical • Optical

11. Electrical Counting • Gain in precision and reproducibility • Lower coefficient of variation and complete a large number of determinations quickly • Cost of the electrical cell-counting equipments ($2500 to over $50,000) • Samples has to be diluted before the count

12. Constant Current Source Vacuum Pump Electrodes 9% NaCl Electrolyte Aperture Tube with Aperture Cell Direction of Flow Container Impedance Principle • Constant current • Insulated chambers • Vacuum pump • Isotonic electrolytes • More on next slide

13. Aperture size is 50-100um “Aperture size: 80 µm for commercial unit” Measure changes in electrical resistance Change in impedance is proportional to individual volume Accurately counts and sizes cells Impedance Principle (Cont’d)

14. Capacitance Principle • Similar idea as the impedance method • Measured in the function of the change in capacitance • However, pulse amplitude generated is not proportional the cell size

15. Outlet Light Source Beam Aperture Photodiode Darkfield stop disk Inlet Darkfield Optical Principle (Cont’d)

16. Darkfield Optical Principle (Cont’d) • The pulse generated by the system is not proportional to the size of the cell • Optical detection is sensitive to size of the dark field stop disk, and the optical magnification • An offset of the parameters will greatly affect the amplitude of the signal

17. Electrical and physical relationships • The pulse height-cell volume relationship can be calculated by using the Maxwell equation:

18. Resistivity of electrolyte • 0.9% NaCl used as the electrolyte • Conductivity of aqueous solutions are usually expressed in Siemens Conductivity (S/cm) = Molarity (mol/L) x ion conductance (SL/cm/eq) x 1 eq/mol • Resistance of the 0.9% NaCl solution is calculated to be 51 Ω/cm

19. Coincidence correction • When a particle is in the aperture, and while the detecting electronics are still busy processing data, the system cannot simultaneously measure another cell

20. Design requirements • Cell sizes that we are measuring vary from 2 μm to 20 μm in diameter • Aperture size of 50-100 μm in diameter will be used • Design of a disposable unit and electronics that can be put in a portable cell counter

21. Cell counter handheld unit

22. Disposable unit (1st design) Aperture To Vacuum

23. Disposable unit (1st design)

24. Images of Disposable unit (1st design)

25. Image of the aperture film under microscope Drilled by laser and measured under electronic microscope ~60um

26. Conclusion • Theory of multi-channel cell counter utilizing the aperture impedance technique have been discussed • Highest resolution available in the industry for particle counting and size distribution • Color or refractive index does not affect results • More design on the disposable unit will be performed and more testing will be done

27. References • [1] Basic Principles in Biology by Y.K.To, Hung Fung Book Co. • [2] Haematology, R.B. Thompson • [3] Kubitschek HE: Counting and sizing micro-organisms with the Coulter counter, in Methods in Microbiology, ed DW Ribbons and JR Norris. London: Academic Press, 1969 • [4] Coulter WH: High speed automatic blood cell counter and cell size analyzer. Presented at the National Electronics Conference, Chicago, October 1956 • [5] Hayes TL: The scanning electron microscope: principles and applications in biology and medicine. Adv Biol Med Phys 12:85, 1968 • [6] Brightfield and darkfield: http://www.wsu.edu/~omoto/papers/Fig1.html • [7] Mansberg HP: Optical techniques of particle counting, in Advances in Automated Analysis, Vol 1. Technicon International Congress. New York: Mediad, 1969 • [8] Hematology; principles and practice. Edited by Charles E. Mengel, Emil Frei, III [and] Ralph Nachman. • [9] http://www.principalhealthnews.com/topic/topic100587682 • [10] http://www.utmem.edu/physpharm/.010.html • [11] Brecher G et al: Evaluation of an electronic red cell counter. Am J Clin Pathol 16:1439, 1956 • [12] Ionic reactions and equilibria. New York : Macmillan, [1967] • [13] http://www.colby.edu/chemistry/CH141B/CH141B.Lab/CH141L4condFall2002.pdf • [14] Practical guide to modern hematology analysers, warren Groner, Elkin Simson, john wiley and sons ltd, 1995

28. Questions