General Approach in Investigation of Haemostasis Lecture

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General Approach in Investigation of Haemostasis
Lecture 9 Coagulation Instruments
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Evaluation of Coagulation tests

• Manual Method
• Coagulometers
• Semiautomated Method.
• Automated Method.

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• 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

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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.

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Semi Automated Method.

• All reagents and samples are added manually by
  the operator.
• Usually contains a device for maintaining'
  constant 37C 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.

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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 37?C
  temperature throughout testing sequence.
• Timers are initiated and clot formation detected
  automatically

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• Manual Method Sem iautomated Method
  Automated Method.

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Methods of Endpoint Detection

• Mechanical
• Optical
• Photo-optical
• Nephelometric
• Chromogenic
• Immunologic
• Electrochemical

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Mechanical

• Two primary methodologies are utilized for
  mechanical detection of clot formation.
• Electromechanical (Impedance) Method.
• Magnetic, Steel Ball Method.

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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.

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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.

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There are two variations of this principle used
in current instrumentation.

• 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.

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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.

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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.

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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 gt600 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.

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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 90-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.

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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 (p-nitroaniline or pNA), which
  has an optical absorbance peak at 405 nm on a
  spectrophotometer.

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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.

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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.

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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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