Blood Cell Counters

• Changes in the normal functioning of an organism are often

accompanied by changes in the blood cell count. Therefore, the
determination of the number and size of blood cells per unit volume
often provides valuable information for accurate diagnosis.
• The percentage of cells in the blood is called the haematocrit value or
packed cell volume (PCV). The majority of the corpuscles in blood are
red blood cells (erythrocytes), others being white blood cells
(leucocytes) and platelets (thrombocytes).
Different methods of cell counting
• Microscopic Method
• Automatic Optical Method
• Electrical Conductivity Method (coulter counters)
Microscopic Method

• The most common and routinely applied method of counting blood

cells even today, particularly in small laboratories, is the microscopic
method in which the diluted sample is visually examined and the cells
counted. Commonly known as the counting chamber technique, it
suffers from several common drawbacks
• Another problem with microscopic counting is that the data gathered
by this measurement is not directly suitable for storage or for further
processing and evaluation.
• Large data analysis will be difficult
Optical Method of counting cells
• The method is based on collecting scattered light from the blood cells and
converting it into electrical pulses for counting.
• A sample of dilute blood (1:500 for white cells and 1:50,000 for red cells) is
taken in a glass container.
• It is drawn through a counting chamber in which the blood stream is
reduced in cross-section by a concentric high velocity liquid sheath.
• A sample optical system provides a dark field illuminated zone on the
stream and the light scattered in the forward direction is collected on the
cathode of a photomultiplier tube.
• Pulses are produced in the photomultiplier tube corresponding to each cell.
• These signals are amplified in a high input impedance amplifier and
fed to an adjustable amplitude discriminator.
• The discriminator provides pulses of equal amplitude, which are used
to drive a digital display.
• Instruments based on this technique take about 30 s for completing
the count.
• An accuracy of 2% is attainable. The instruments require about 1 ml
of blood sample.
Electrical Conductivity Method
• Blood cell counters, operating on the principle of conductivity change, which
occurs each time a cell passes through an orifice, are generally known as Coulter
Counters.
• The underlying principle of the measurement is that blood is a poor conductor of
electricity whereas certain diluents are good conductors.
• For a cell count, therefore, blood is diluted and the suspension is drawn through a
small orifice.
• By means of a constant current source, a direct current is maintained between
two electrodes located on either side of the orifice.
• As a blood cell is carried through the orifice, it displaces some of the conductive
fluid and increases the electrical resistance between the electrodes.
• A voltage pulse of magnitude proportional to the particle volume is thus produced.
• The resulting series of pulses are electronically amplified, scaled and displayed on
a suitable display.
• To achieve optimum performance and to enable the relationship of
change in resistance with volume of the cell to hold good, it is
recommended that the ratio of the aperture length to the diameter of
the aperture should be 0.75:1, i.e. for an orifice of 100 μ diameter the
length should be 75 μ.
COULTER COUNTERS
• A glass measuring tube 'C' provided with an aperture 'A' is immersed into the
suspension.
• The pressure difference created between the two sides of the aperture draws
the suspension to flow through the aperture.
• This pressure difference is generated by a simple mechanical pump consisting
of a syringe, a relay and other parts.
• A constant current is normally passed between the electrodes E1 and E2.
• Therefore, the electric resistance of the liquid measured between these two
electrodes changes rapidly when a particle having electric conductance
differing from the conductance of the electrolyte passes through the aperture
• . This results in the generation of a voltage pulse, which is amplified in a
preamplifier of high gain and low noise level.
• The output signal of this stage goes to a discriminator, which compares the
amplitude of the pulse arriving at its input with the preset triggering level.
• If the input signal exceeds the triggering level, the discriminator gives out a
pulse of constant shape and amplitude.
• These pulses go to a counting circuit for the display of the measured
parameter.
• Fig. 16.5 shows the sequence of building up the pulse in terms of increase
in resistance at different positions of the cell with respect to the orifice.
• To enable the instrument to count only those pulses, which fall within
certain preset size limits, the threshold facility is required.
• The threshold is also necessary to enable the instrument to ignore
any electronic noise, which may be present in the system.
• The lower threshold sets an overall voltage level, which must be
exceeded by a pulse before it can be counted. The upper threshold
will not allow pulses to be counted which exceed its preset level.
• The measuring tube C is provided with a third electrode E3 which
helps to monitor the suction of a limited volume of the suspension.
• When the liquid level reaches E3, the pump is changed over from the
suction phase to the pressure phase.
• The counting process also occurs during the time the electrolyte is
forced out of the measuring tube.
• After the liquid looses contact with the electrode E1, the counting
automatically stops and the unit becomes ready once again for the
operation.
AUTOMATIC RECOGNITION AND
DIFFERENTIAL COUNTING OF CELLS
• Along with the automated instruments for obtaining the erythrocyte,
leucocyte and platelet counts, there has been a considerable interest in
developing automated techniques for identifying and counting the
different types of cells within a given class. Examples of this could be
the immature red cell count, the differential leucocyte count and the
recognition of normal versus malignant cells in other cell types.
• If the cell is identified as one of the six normal cell types, it is counted
otherwise it is shown as a suspect cell to be identified by the operator.
The same field is searched again for other nucleated cells. If no other is
found, then the stage drive is incremented and the electronic search
resumes in the new field presented to the system.
 Diff-3 System
• Counts and differentiates seven important categories of red blood
cells (erythrocytes); three based on size, two on colour, one on shape
and one covering red cells with nucleii (nucleated red cells).

• Enumerates white blood cells (leucocytes) and differentially

classifies them into the 10 most significant medical categories and
estimates their total number.

• Surveys platelets (thrombocytes) cell size and sufficiency.

• Operation read from Khandpur R.S, “Handbook of Biomedical
Instrumentation”, Tata McGraw-Hill, New Delhi,
FLOW CYTOMETRY
• In a flow cytometry system, microscopic particles are suspended in a
stream of fluid and individual cells are characterized simultaneously
for physical and/or chemical properties.
• Fluorescent chemicals found in cells or attached to cells are analyzed,
as they flow through a tube and are passed in front of a light source.
• The light that is scattered from each cell is measured with a
photodiode and by analyzing the changes in brightness at each
detector, it is possible to determine various types of information
about the physical and chemical structure of each individual cell.
• The cells to be counted are first stained with proteins that fluoresce in
response to certain wavelengths of light. These proteins are designed
to attach only to certain cells. In some cases, cells are not stained, as
they have native proteins that fluoresce. The cells are forced down a
tube whose pore size has been set to allow only a specific range of
cell sizes to pass. These cells flow past one to four lasers with
different wavelengths. When passing through the laser beam, the
proteins will fluoresce at a longer wavelength than the lasers.
• Photodetectors capture these wavelengths and measure the scattered
light from the original source. The different wavelengths allow the
instrument to determine the size and type of cell. The output of the
photodetectors is amplified and used to drive the ADCs. Typically,
there is one ADC per channel. All of the data is processed by a digital
signal processor (DSP), and histograms are generated showing the
types of cells counted. Once the cells pass by the laser, they are
charged by a charging ring. The amount of charge is determined by
the type of cell. Deflection plates then sort the cells into different test
tubes for further analysis.