General Approach of Haemostasis Prothrombin Time (Continue) Lecture 5:

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 1.0Introduction

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. Calculating the ISI

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

 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 = Example:

 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. Effects of Sensitivity Index: Patient PT [s] Thromboplastin 1 [ISI 1.0] Thromboplastin 2 [ISI 1.5] Thromboplastin 3 [ISI 2.0]

 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. Local Calibration

Reporting of Result  Time (Seconds)  Ratio (PT patient /PT control ) PT r  Activity Percentage PT p  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.liver disease In patients on oral anticoagulant therapy, only INR standardize PT results.anticoagulant 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).

 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. PT Activity percentage 1:801:401:201:10 Pooled Plasma 12.5%25%50%100%Percentage

Next Lecture: Coagulation Instruments