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Prothrombin Time
Prothrombin time (PT) was one of the first generated in vitro tests of the hemostasis system. It measures the time to form a fibrin clot in platelet-poor plasma stimulated with high amount of tissue factor (TF) and anionic phospholipid at the optimal concentration of calcium. The clotting is initiated by phospholipid-bound TF binding factor VII or VIIa (activated form of factor VII [FVII]). Under the test conditions, FVIIa can directly activate FX, which in turn initiates conversion of prothrombin to thrombin with slow kinetics. The small amount of thrombin in the initiation phase generates activated FV. FVa greatly accelerates FXa protease activity toward prothrombin, allowing the production of enough thrombin to rapidly convert fibrinogen into fibrin, forming a clot. Clot formation can be detected either by measuring changes in plasma viscosity via an electromechanical device or plasma absorbance via photooptical means. The time to clot is reported in units of seconds.
Since the test’s invention over 75 years ago, its significance has much expanded, as it has found new uses in anticoagulant drug monitoring. Today it is likely the most widely ordered laboratory coagulation test, with over 800 million PT tests performed annually in the world. The test is sensitive to multiple plasma coagulation factors and suffers from fewer interferences than activated partial thromboplastin time (PTT). This makes it the test of choice for assessing plasma coagulation factor status in situations where all the factors are affected similarly, including trauma, disseminated intravascular coagulopathy (DIC), and surgery. Additionally, high levels of FVIII that shorten PTT do not affect PT. Because FVIII elevations are often present in liver disease, PT is more sensitive to compromise synthetic function of the liver. PT is still the test of choice to monitor treatment with vitamin K synthesis inhibitors (warfarin). Modified PT-based tests are used to measure concentrations of factors II, V, VII, and X. Some commercial tests use PT-type activation for measuring anticoagulants protein C and S activities, as well as activated protein C resistance.
Quick and Owren Prothrombin Time
Two variants of the PT test are currently in use. In most countries, the earlier version of the test based on the Quick method dominates. In the Quick-type PT test, TF and phospholipid are added to decalcified plasma with a final test plasma dilution of 1:3. This variant of the test assesses concentrations of vitamin K-dependent factors II, VII, and X, and additionally, non–vitamin K-dependent FV and fibrinogen.
The Owren-type PT test is now used predominantly in the Nordic countries, Benelux, and Japan. This test was designed specifically to monitor vitamin K-dependent factors and is insensitive to FV and fibrinogen concentration in the test plasma. In the Owren-type PT method, bovine plasma depleted of vitamin K-dependent factors is added together with calcium, phospholipid, and TF. The bovine plasma provides a source of fibrinogen and FV, making the test sensitive only to concentration of vitamin K-dependent factors II, VII, and X. The test plasma is diluted 1:21.
International Normalized Ratio
Many different sources of TF and phospholipid used in commercial PT reagent preparations pose a significant challenge to standardizing the monitoring of oral anticoagulants using PT. There is considerable variation in the sensitivity of various PT formulations to the concentration of the vitamin K-dependent factors. Therefore, taking a simple ratio of the test PT to the mean of normal PT would not produce comparable results when different reagents are used. To harmonize the treatment monitoring of oral anticoagulants, each reagent lot is assigned an international sensitivity index (ISI) based on an international standard. The ISI is calculated by comparing PTs obtained from a given reagent and instrument combination to that of a reference reagent obtained by using a manual tilt-tube method. This comparison is done using plasmas deficient in vitamin K-dependent factors and is usually performed by the reagent vendor. It is preferable that the ISI calibration is performed for specific combinations of reagent and instrument. If generic ISI (reagent only) is used, local ISI calibration verification is now mandatory (see Clinical Laboratory Standards Institute [CLSI] document H54-A). The international normalized ratio (INR) can be calculated as follows:
It should be noted that ISI calibration is performed using only vitamin K-deficient plasmas; the sensitivity determination is only relevant to detection of vitamin K deficiency or to monitoring warfarin therapy. Other conditions that prolong PT, such as liver failure or DIC, involve decreases in FV and alterations in quantity and/or quality of fibrinogen. Sensitivity to these changes is not assayed during ISI calibration. It has been shown that the use of INR is the single largest contributor to interinstitutional variation of the model of end-stage liver disease score used for liver transplant priority list. Determining ISI using plasma from liver disease patients has been shown to dramatically improve INR variation in liver failure. Unfortunately, liver disease ISI determination is not routinely performed. Further details of PT use for monitoring oral anticoagulant therapy are provided in Chapter 156 .
Test Limitations
Common preanalytical and analytical variables that can limit the usefulness of PT for monitoring coagulation factor levels are listed below.
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