Practical Procedure

Prothrombin Time
By
Mac-Kadicou K. ORJI
(B.MLS; M.Sc; PhD-IV; IFBA-Certified, MNiBSA; AMLSCN; AAAS; MISM)
 Overview
• When a body tissue is injured and begins to
bleed, it initiates a sequence of clotting
factor activities, known as the coagulation
cascade, leading to the formation of a blood
clot.

• This cascade is comprised of three

pathways:
• extrinsic, intrinsic, and common.
 Overview contd.
• The diagnostic investigations commonly
used to evaluate coagulation disorders:

• Prothrombin Time (PT) which measures

the integrity of the extrinsic system as well
as factors common to both systems and
• Partial Thromboplastin Time (PTT),
which measures the integrity of the intrinsic
system and the common components.
 Intro/Background
• Blood clotting is a function of plasma.
• It depends upon the orderly interaction of a
group of plasma proteins sequentially activated
following vascular injury with some
phospholipid which may come from either
damaged tissue or platelets and some Calcium
ions.
• The final stages include the formation of
thrombin, which then converts: soluble plasma
protein fibrinogen --------> insoluble fibrin.
 Intro/Background contd.
• Another factor converts the fibrin into a cross-
linked polymer which stabilizes the platelet
plug and traps RBCs in the meshwork to form
the actual blood clot.
• Depending on the type of vascular damage or
abnormality, clotting can be initiated and
proceed according to two different cascading
pathways: the intrinsic -initiated by contact
with an abnormal or foreign surface, or the
extrinsic -initiated by exposure to tissue factors
 The Cascade
• In the extrinsic pathway of blood coagulation,
a tissue factor is produced after injury, and
released from damaged cells.

• The tissue factor interacts with factor VIIa to

activate factor X to Xa.
Factor Xa then acts on prothrombin according
to the common pathway of coagulation.
The Complete Cascade
 Title
• Prothrombin Time
• PT
 Aim/Objective

• To estimate the time (measured

in seconds) taken for plasma to
clot after addition of CaCl2
reagent and thromboplastin.
 Principle
• Citrated plasma is able to continue
the coagulation process via the
Extrinsic and common pathway
upon addition of CaCl2 reagent in
the presence of thromboplastin.
 Apparatus/Equipment/Materials
• Serum derived from Citrated blood
• Automatic Pipette
• Test Tubes
• PT Reagent
• Water bath
• Timer
 Methodology/Procedure
• Pipette 100µl (0.1ml) of citrated
plasma into test tube
• Incubate for 2 minutes at 37o C
• Add 200µl (0.2ml) of pre-warmed
thromboplastin calcium reagent and
start the stop watch
• Observe for clotting
Schematics of PT
 Observations/Results
• Normal Range = 11 – 16seconds

• The prothrombin test specifically

evaluates the presence of factors VII,
V, and X, prothrombin, and
fibrinogen.
 Interpretation/Conclusion
• A prolonged prothrombin time indicates a deficiency
in any of factors VII, X, V, prothrombin, or
fibrinogen.
• It may mean that the patient has a vitamin K
deficiency (vitamin K is a co-factor in the synthesis of
functional factors II (prothrombin), VII, IX and X)
• It may mean that the patient has a liver disease (the
liver is the site of synthesis of the plasma protein
factors).
• The prothrombin time of patients receiving a vitamin
K-competing coumarin drug such as warfarin
(anticoagulation therapy used in DVT) will also be
prolonged, usually in the range of one and one half to
two times the normal PT time.
 INR & ISI
• The clotting time in seconds is converted to the
International Normalized Ratio (INR), usually by
reference to a table provided by the manufacturer of
the reagent or from the formula.
• INR = (PT Patient ÷ PT Control)ISI* i.e. prothrombin
ratio raised to the power of the ISI

• *International Sensitivity Index:

• This figure is provided by the manufacturer of the
thromboplastin reagent.
• To obtain the INR, calculate the prothrombin ratio,
log the ratio, multiply by the ISI, and antilog the
result.