General Approach of

Haemostasis
Lecture 5:

Prothrombin Time
(Continue)
Introduction
The Prothrombin Time (PT) will vary with the
type of thromboplastin (e.g. rabbit, human,
bovine etc) used in the assay.
This difference in sensitivities is known as the
sensitivity index.
Individual thromboplastins can be calibrated
against an international WHO reference
thromboplastin (International Reference
Preparation or IRP) to assign them an
International sensitivity index or ISI.
The first WHO reference thromboplastin was
assigned an ISI of 1.0
Calculating the ISI
The calibration of a thromboplastin must be against a
reference thromboplastin of the same species e.g.
human against human, rabbit against rabbit etc.
Prothrombin Times are performed in duplicate for each
thromboplastin and the mean for each pair of tests
derived.
Tests are performed on 20 normal donors not on
anticoagulants and 60 patients who have been on oral
anticoagulant treatment for at least 6 weeks.
If there is more than a 10% difference in the clotting
times between duplicate samples, the tests on that
plasma sample should be repeated.
The mean of each pair of PT results are plotted on
double-log paper with the reference sample on the Y
axis and the test plasma on the X-axis.
A best fit line is drawn with points above the highest
recorded PT and the lowest PT. The slope of the line
is calculated and this represents the ISI
Example:
 Distance A to C = 125mm and distance B to C =
110mm

 So the ISI of the test thromboplastin is calculated

from the formula:

 So if the ISI of the Reference Thromboplastin is 1.1

and the ISI derived from the Slope is 1.14, the ISI of
the Test Thromboplastin is 1.1 x 1.14 = 1.25.
Effects of Sensitivity Index:
 Thromboplastins should be chosen with an ISI close
to 1.0
 The next table show this by Using a thromboplastin
with an ISI of 1.0 the PT can range from 15s - 35s but
the INR is still in the therapeutic range. In contrast if
the ISI of the thromboplastin is 2.0 an increase in the
PT from 15s to 30s results in an INR outwith the
therapeutic range.

Patient PT [s] Thromboplastin 1 Thromboplastin 2 Thromboplastin 3

[ISI 1.0] [ISI 1.5] [ISI 2.0]
15 1.25 1.39 1.54
20 1.67 2.15 2.78
25 2.08 3.00 4.3
30 2.5 3.95 6.25
35 2.91 4.98 8.51
Local Calibration
 Use of different coagulometers for the PT and the
differing methods of end-point detection can lead to
significant variations in PT. For these reasons, a local
calibration of thromboplastins is recommended.
 This involves testing a set of plasma samples with
known INRs with the a laboratory-specific
thromboplastin and on the coagulometers which will
be used to derive the PT.
Reporting of Result
 Time (Seconds)
 Ratio (PTpatient/PTcontrol) PTr
 Activity Percentage PTp
 INR
The International Normalised Ratio
 The International Normalised Ratio (INR) is the PT
ratio of a test sample compared to a normal PT (of
20 normal donors) corrected for the sensitivity of the
thromboplastin used in the test.
 It is the value for the Prothrombin Time Ratio which
has been obtained using the first WHO Reference
Thromboplastin with an ISI of 1.0

 Example; for patient on warfarin and his PT 23 and

the Normal PT is 12 with ISI 1.2
 The previous table illustrates a number of patients
with varying PTs and in whom the INR was
calculated using three Thromboplastins with varying
ISIs.
 The simplest way to calculate the INRs is to use an
electronic calculator!
PT Activity percentage
The result reported as percent activity of normal
instead of seconds as the unit of measurement of the
Quick test.
A study show that the results were expressed in PTp
but not INR, PTs and PTr is valid for comparison of
patients with liver disease.
In patients on oral anticoagulant therapy, only INR
standardize PT results.
Pooled, normal plasma that is assumed to be 100% of
normal activity is diluted with any variety of diluents
(barium-sulfate-adsorbed human plasma, barium-
sulfate-adsorbed beef plasma, 0.85% saline,
imidiazole-saline buffer).
PT Activity percentage
 Prothrombin activity assayed by on stage assay.
 To obtain PTp expression, a saline dilution curve
was constructed with normal pool plasma and
the patient result was expressed as the
percentage of normal plasma yielding the same PT in
seconds.

Pooled Plasma 1:10 1:20 1:40 1:80

Percentage 100% 50% 25% 12.5%

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Next Lecture: Coagulation Instruments