Dr.

Marwa IBRAHIM KHEDR, MD

Lecturer of Medical Biochemistry
Clinical Pathology specialist

Dr. MARWA IBRAHIM KHEDR, MD

Physiology o f Blood C l o t t i n g

Dr. MARWA IBRAHIM KHEDR, MD

8
HEMOSTASI S
1. VASCULAR PHASE
2. PLATELET PHASE
3. COAGULATION PHASE
4. FIBRINOLYTIC PHASE

Dr. MARWA IBRAHIM KHEDR, MD

 Lab Tests
•CBC-Plt
•BT,(CT)
BV Injury •PT
Tissue •PTT
Neural Factor

Blood Vessel Platelet Coagulation

Constriction Aggregation Cascade
Primary hemostatic plug
Reduced Platelet
Activation Fibrin
Blood flow formation
Plt Study
Morphology
Stable Hemostatic Plug Function
Antibody
Dr. MARWA IBRAHIM KHEDR, MD
Pri m ary Hem os tas i s
◦ Blood vessel contraction
◦ Platelet Plug Formation
Secondary Hemostasis
◦ Activation of ClottingCascade
◦ Deposition &Stabilization of Fibrin
Tertiary Hemostasis
◦ Dissolution of Fibrin Clot
◦ Dependent on Plasminogen Activation

Dr. MARWA IBRAHIM KHEDR, MD

 C
O
A
G
U
L
A
T
I
O
N
Dr. MARWA IBRAHIM KHEDR, MD
Dr. MARWA IBRAHIM KHEDR, MD
 PROVIDES ASSESSMENT OF PLATELET
COUNT AND FUNCTION

NORMAL VALUE
2-8 MINUTES

Dr. MARWA IBRAHIM KHEDR, MD

Robbins Basic Pathology , 10th Edition, Elsevier 15
 PROTHROMBIN TIME
Measures Effectiveness of the Extrinsic Pathway
Mnemonic - PT

NORMAL VALUE
10-15 SECS

Dr. MARWA IBRAHIM KHEDR, MD

Robbins Basic Pathology - 10th Edition - Elsevier
 INR

Dr. MARWA IBRAHIM KHEDR, MD

 INR

Dr. MARWA IBRAHIM KHEDR, MD

 Measures Effectiveness of the Intrinsic
Pathway
Mnemonic - PTT

NORMAL VALUE
25-40 SECS

Dr. MARWA IBRAHIM KHEDR, MD

Robbins Basic Pathology - 10th Edition - Elsevier

 THROMBIN TIME
Time for Thrombin To Convert
Fibrinogen Fibrin
A Measure of Fibrinolytic Pathway

NORMAL VALUE
9-13 SECS

Dr. MARWA IBRAHIM KHEDR, MD

Robbins Basic Pathology - 10th Edition - Elsevier
Dr. MARWA IBRAHIM KHEDR, MD
Dr. MARWA IBRAHIM KHEDR, MD
Screening tests:
◦ Bleeding.T - 10m. Platelet & BV function
◦ Prothrombin.T – Extrinsic, aPTT – Instrinsic
◦ Thrombin.T – common path. (DIC)

Specific tests:
◦ Factor assays – hemophilia.
◦ Tests of thrombosis – TT,
◦ Platelet function studies:
Adhesion, Aggregation, Release tests.
◦ Bone Marrow study

Dr. MARWA IBRAHIM KHEDR, MD

VESSEL DEFECTS
?
PLATELET DISORDERS
FACTOR DEFICIENCIES
OTHER DISORDERS
?

Dr. MARWA IBRAHIM KHEDR,

 Classification:
• Disorders of Blood vessels
• Scurvy, senile purpura, Henoch-Schonlein syndrome.

• Disorders of Coagulation
• Extrinsic, intrinsic, combined.

• Disorders of Platelets
• Thrombocytopenia ITP, TTP, HUS, DIC.
• Aspirin therapy, Thrombasthenia,

• Other disorders
• Post transfusion purpura.

Dr. MARWA IBRAHIM KHEDR, 22

Coagulation tests
• Manual Method
• Coagulometers
1. Semi-automated Method.
2. Automated Method.

Dr. MARWA IBRAHIM KHEDR, MD

Coagulation tests
• semi-automated equipment are based
on:
•Optical principle
•Mechanical principles to detect fibrin.

Dr. MARWA IBRAHIM KHEDR, MD

Coagulation tests
• Instruments have become common in
modern laboratories.
•Today, new equipment connected to
specific data processing systems can
undertake Clotting, Chromogenic, and
immunological tests.

Dr. MARWA IBRAHIM KHEDR, MD

Coagulation tests (Manual Method)
•All reagents and samples are added manually by
the operator.
•Temperature is maintained by a waterbath or
heat block
•Most often using a stopwatch.

Dr. MARWA IBRAHIM KHEDR, MD

Coagulation tests (Manual Method)
•All reagents and samples are added manually by
the operator.
•Temperature is maintained by a waterbath or
heat block
•Most often using a stopwatch.

Dr. MARWA IBRAHIM KHEDR, MD

Coagulation tests (Semi auto 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.

Dr. MARWA IBRAHIM KHEDR, MD

Coagulation tests (Semi auto Method)

Dr. MARWA IBRAHIM KHEDR, MD

Coagulation tests (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 37oC temperature throughout
testing sequence.
• Timers are initiated and clot formation
detected automatically.
Dr. MARWA IBRAHIM KHEDR, MD
Methods of Endpoint Detection

• Mechanical
• Optical
• Nephelometric
• Chromogenic
• Immunologic
• Electrochemical

Dr. MARWA IBRAHIM KHEDR, MD

Mechanical
Two primary methodologies are utilized
for mechanical detection of clot formation.

• Electromechanical (Impedance)
Method.
• Magnetic, Steel Ball Method.

Dr. MARWA IBRAHIM KHEDR, MD

Mechanical 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.
Dr. MARWA IBRAHIM KHEDR, MD
 Electromechanical method
using moving electrical probe.
Dr. MARWA IBRAHIM KHEDR, MD
Mechanical Steel ball based 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.

Dr. MARWA IBRAHIM KHEDR, MD

 Steel Ball based Method (A)
Change in range of motion of magnetic steel ball
Dr. MARWA IBRAHIM KHEDR, MD
 Steel Ball based Method (B)
break-in contact of steel ball with
the magnetic sensors
Dr. MARWA IBRAHIM KHEDR, MD
 Photo-Optical
• 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.

Dr. MARWA IBRAHIM KHEDR, MD

 Photo-Optical
• 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.

Dr. MARWA IBRAHIM KHEDR, MD

 Optical method
(turbidimetry and nephelometry)
Dr. MARWA IBRAHIM KHEDR, MD
 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.

Dr. MARWA IBRAHIM KHEDR, MD

 Immunologic
• 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.
•
Dr. MARWA IBRAHIM KHEDR, MD
 Immunologic
• 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.
Dr. MARWA IBRAHIM KHEDR, MD
 Electrochemical
INRatio Meter (Hemosense)
• 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.
•

Dr. MARWA IBRAHIM KHEDR, MD

Electrochemical

Dr. MARWA IBRAHIM KHEDR, MD

Electrochemical

Dr. MARWA IBRAHIM KHEDR, MD

Dr. MARWA IBRAHIM KHEDR, MD