Cell counters

Prof.S.Vidhya
 Blood cell sizes and their normal
ranges

Blood cell type Sizes Normal Ranges

(um) (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
 Diseases of the Blood
Cell Type Increase count
WBC • Infectious diseases
• Inflammatory disease
• Severe emotional
• Physical stress
• Tissue damage
RBC • Renal tumor
• Iron overload in organs
Platelet • Renal disease
• Infection or inflammation
Decrease count
• Bone marrow failure
• Presence of toxic substance
• Disease of the liver/spleen
• Radiation
• Anemia
• Chronic inflammation
• Anemia
• Bone marrow failure
• Uremia
• Liver disease
 Blood cell counter
• The blood cell counter count the number
of RBC or WBC per unit of volume of
blood using either of two method:

– Electrical method called aperture impedance

change
– Optical method called flow cytometry
 Aperture impedance change
• When blood is diluted in the proper type of
solution, the electrical resistivity of blood
cells (ρc) is higher then the resistivity of the
surrounding fluid (ρf)
• By contriving a situation in which these
resistivities can be differentiated from each
other, we can count cells
 Aperture impedance change
Blood cell sensing
• The sensor consist of a two-chamber vessel
in which the dilute incoming blood is on one
side of barrier, and the waste blood to be
discarded is on the other
• A hole with a small diameter (50µm) is placed
in the partition between the two halves of the
cell
• Ohmmeter measure the change on the
resistance when the blood cell pass the
aperture
Blood cell sensing
 Coulter Counter
• Constant current source (CCS) and
voltage amplifier replace the ohmmeter
• RA is the resistance of the aperture and will
be either high or low, depending on
whether or not the blood cell is inside the
aperture.
• Amplifier convert the current pulse to
voltage pulse
Schematic of Coulter Counter
Electrical and physical relationships

• The pulse height-cell volume relationship

can be calculated by using the Maxwell
equation:

(i ⋅ ρ 2 )
∆Voltage = ∆V 2
A
 Coulter counter- Impedance
Principle
• Constant current
• Insulated chambers
Constant
• Vacuum pump Current Source Vacuum Pump
• Isotonic electrolytes

Electrodes 9% NaCl
Electrolyte

Aperture
Tube with
Cell Aperture
Direction of
Container Flow
Coulter counter- 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
 Flow cytometry cell counters
optical flow cytometry sensing

• The optical cytometry sensor consists of a

quartz sensing sheath of PBS (Phosphate
buffered saline) designed with a
– hydrodynamic focusing region
– cell path region that passes only a single cell
at time.
• Focusing is done by decreasing the
diameter of the aperture.
• Light source is (He-Ne) Laser
 Flow cytometry cell counters
optical flow cytometry sensing

• Two Photodetectors (photosensors)

– Photodetector A detects forward scattered light
– Photodetector B detects orthogonal scattered light
• Scattered and emitted light from cells and particles are converted to
electrical pulses by optical detectors. Collimated (parallel light
waveforms) light is picked up by confocal lenses focused at the
intersection point of cells and the light source. Light is send to
different detectors by using optical filters.
• blood sample enters the analyzer
– Optical counter → WBC count
– Colorimeter → hemoglobin
– Optical flow sensor → RBC count
Schematic