Multi-channel Cell Counter Utilizing The Aperture Impedance

Technique

Aaron Lee & Dr. Ash M. Parameswaran

Simon Fraser UniversitySchool of Engineering SciencesBurnaby, B.C. Canada V5A 1S6

Email: cleek@sfu.ca

This work is sponsored by Brain Insights, California

Overview

1. Introduction

2. Centralized approach

3. Clinical facts

4. Techniques of cell counting

5. Electrical and physical relationships

6. Disposable unit design

7. Conclusion

Thesis Concentration

Construction, modeling and testing of the disposable unit and the electronics

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

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 timePatient has to visit another time

Commercial blood cell counter

18 cell sizes result and histograms

Dimensions: 37x47x38(cm)

Weight: 18 kg net Power: AC No portable blood

counter in the market

Our challenges

Shortens the turn around timeReduce the cost so clinics can afford to

own the blood cell counterMiniaturize the testing equipmentMaintain or improve accuracy

Blood cell type Sizes(um)

Normal Ranges (per mm3)

-- -- Male Female

Red blood cell 6-10 4.5-6.5 M 3.9-5.6 M

White blood cell 10-20 4.5-11 k 4.5-11k

Platelets 2-4 150-350 k 150-350 k

Blood cell sizes and their normal ranges

Diseases of the Blood

Cell Type Increase count Decrease count

WBC Infectious diseases Inflammatory disease Severe emotional Physical stress Tissue damage

Bone marrow failure Presence of toxic substance Disease of the liver/spleen Radiation

RBC Renal tumor Iron overload in organs

Anemia Chronic inflammation

Platelet Renal disease Infection or inflammation

Anemia Bone marrow failure Uremia Liver disease

Cell count techniques

ElectricalOptical

Electrical Counting

Gain in precision and reproducibility Lower coefficient of variation and complete

a large number of determinations quicklyCost of the electrical cell-counting

equipments ($2500 to over $50,000) Samples has to be diluted before the

count

Impedance Principle Constant current Insulated chambers Vacuum pump Isotonic electrolytes More on next slide

Container

ApertureTube with ApertureCell

9% NaCl Electrolyte

Vacuum PumpConstant

Current Source

Electrodes

Direction of Flow

Impedance Principle (Cont’d)

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

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

Darkfield Optical Principle (Cont’d)

Inlet

Outlet

Darkfield stop disk

Light SourceBeam

AperturePhotodiode

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

Electrical and physical relationships

The pulse height-cell volume relationship can be calculated by using the Maxwell equation:

22 )(

A

iVVoltage

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

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

Activity

RateCountRawratecountCorrected

1

____

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

Cell counter handheld unit

Disposable unit (1st design)

To Vacuum

Aperture

Disposable unit (1st design)

Images of Disposable unit (1st design)

Image of the aperture film under microscope

~60um

Drilled by laser and measured under electronic microscope

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

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

Questions