General Approach in
Investigation of Haemostasis
Lecture 10:
Coagulation Instruments
Evaluation of Coagulation tests:
 Manual Method:
 Coagulometers:

Semiautomated Method.

Automated Method.

Manual methods were used at the begining,
then semi-automatic equipments appeared
based on photometric or mechanical principles
to detect fibrin. More recently, fully automated
instruments have become common in modern
laboratories.

Today, new equipment connected to specific
data processing systems can undertake clotting,
Chromogenic, and immunological tests.

Automation has contributed to improvements
in standardization and facilitating tests –
improve lab efficiency and repertoire
Manual Method

All reagents and samples are added
manually by the operator.

Temperature is maintained by a waterbath
or heat block;

May require external measurement by
operator, most often using a stopwatch.
Semi Automated Method.

All reagents and samples are added manually
by the operator.

Usually contains a device for maintaining'
constant 37°C temperature, Analyzer may or
may not internally monitor temperature .

Has mechanism to automatically initiate timing
device upon addition of final reagent and
internal mechanism for detecting Clot
formation.
Automated Method.

All reagents are automatically pipetted by the
instrument. Samples may or may not be
automatically pipetted.

Contains monitoring devices and internal
mechanism to maintain and monitor constant
37οC temperature throughout testing sequence.

Timers are initiated and clot formation detected
automatically

Manual Method
Sem iautomated Method
Automated Method.
Methods of Endpoint Detection

Mechanical

Optical

Photo-optical

Nephelometric

Chromogenic

Immunologic

Electrochemical
Mechanical
Two primary methodologies are utilized for
mechanical detection of clot formation.

Electromechanical (Impedance) Method.

Magnetic, Steel Ball Method.
Electromechanical Method.

When coagulation process takes place, the concentration of
clotting factors (charges) and inorganic ions will change
along the time and the measured impedance or conductance
will be also changed correspondingly at the same time.

During the reaction, one probe moves in and out of the
solution at constant intervals. The electrical circuit between
the two probes is not maintained as the moving probe rises
in and out of the solution.

When a clot (fibrin) is formed in the solution, the fibrin
strands maintain electrical contact between the two probes
when the moving probe leaves the solution, which stops the
timer.
Magnetic, Steel Ball Method

Mechanical clot detection involves monitoring
the movement of a steel ball within the test
solution using a magnetic sensor. As clot
formation occurs, the movement of the ball
changes, which is detected by the sensor.
There are two variations of this principle used
in current instrumentation.
1.
2.
*A change in the movement of the steel
ball may be detected when there is
increased viscosity of the test solution,
changing its range of motion,
*Or by a break in contact with the
magnetic sensors when the steel ball
becomes incorporated into a fibrin clot as
the cuvette rotates.
Photo-optical Method

Detection of clot formation measured by a change in OD
of a test sample is the basis of photo-optical
instrumentation, which is also known as turbidometric
methodology:
 When a light source of a specified wavelength is passed through a
test solution (plasma), a certain amount of light is detected by a
photodetector or photocell located on the other side of the
solution.
 The amount of light detected is dependent on the color and clarity
of the plasma sample and is considered to be the baseline light
transmission value.
Photo-optical
Method


When soluble fibrinogen begins to polymerize into a
fibrin clot, formation of fibrin strands causes light to
scatter, allowing less light to fall on the photodetector
(i.e., the plasma becomes more opaque, decreasing the
amount of light detected.
When the amount of light reaching the photodetector
decreases to an exact point from the baseline value as
predetermined by the instrument, this change in OD
triggers the timer to stop, indicating clot formation.
Nephelometric



Quantifying plasma proteins based on the specific reaction
of the protein being measured with highly specific antisera.
Precipitants are antigen-antibody complexes, which show
up in solutions as turbidity and scatter incident light.
The nephelometer uses a light emitting diode at a high
wavelength (usually >600 nm) to detect variations in light
scatter as antigen-antibody complexes are formed. When the
light rays encounter insoluble complexes such as fibrin
strands, they are scattered in both forward (l80-degree) and
side (90-degree) angles.

Nephelometric
Instruments employing this type of measuring system detect
the amount of agglutination of particles by reading the
increasing amount of light scattered at a 70-degree angle as
agglutinates are formed. The timer is triggered to stop when
the amount of light scatter reaches a specific predetermined
level.
 This method of endpoint detection is in contrast to the
photo-optical systems, which sense decreased light
transmission at 180 degrees due to the opaqueness of the
sample in a cuvette when fibrin is formed.
Chromogenic
 Chromogenic,
or amidolytic, methodology is based on
the use of a specific color-producing substance known
as a chromophore. The chromophore normally used in
the coagulation laboratory is para-nitroaniline (pnitroaniline or pNA), which has an optical absorbance
peak at 405 nm on a spectrophotometer.
Immunologic Method



Immunologic assays are based on antigen-antibody
reactions.
Latex Microparticles are coated with a specific
antibody directed against the analyte (antigen) to be
measured.
A beam of monochromatic light is then passed
through the suspension of microlatex particles. When
the wavelength of light is greater than the diameter
of the particles in suspension, only a small amount of
light will be absorbed by the particles.
Immunologic
Method



When the latex microparticles coated with specific
antibody come in contact with the antigen present in
the solution, the antigen attaches to the antibody and
forms bridges between the particles, causing them to
agglutinate.
As the diameter of the agglutinates becomes larger
and closer to the wavelength of the monochromatic
light beam, the greater the amount of light that is
absorbed.
The increase in light absorbance is proportional to the
size of the agglutinates, which, in turn, is
proportional to the antigen level present in the
sample, which is read from a standard curve.
Electrochemical
INRatio Meter (Hemosense)
Near Patient Testing Devices
The INRatio single-use test strip is made of
laminated layers of transparent plastic. Each test
strip has a sample well where blood is applied,
three channels through which the blood sample
flows to reach the testing areas, reagents to start the
coagulation process, and electrodes that interface
with the INRatio meter. The device detects a change
in electrical resistance when blood clots.
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