Ecarin Clotting Time
The Ecarin Clotting Time [ECT] is a meizothrombin generation test that can be used to measure the activity of the direct thrombin inhibitors such as r-hirudin (lepirudin) but is likely to be useful with the introduction of the new oral direct thrombin inhibitors. Lepirudin is no longer available in the UK.
Ecarin is a highly purified metalloprotease isolated from the venom of the saw-scaled viper Echis carinatus and a specific activator of prothrombin. Prothrombin is converted in a series of steps to thrombin and ecarin specially cleaves prothrombin at Arg323-Ile 324 to generate meizothrombin an intermediate which is proteolytically active. Meizothrombin is inactivated by hirudin but is unaffected by heparin (as the heparin-antithrombin complex cannot inhibit meizothrombin due to steric hindrance.
For the Activated Clotting Time [ACT] there is no linear correlation between the plasma hirudin levels and the clotting time. For the APTT a linear correlation exists only for plasma hirudin levels up to 1µg/ml depending upon the APTT reagents used. With increasing hirudin concentrations there is no further prolongation of the APTT and so the APTT underestimates the true anticoagulant effect of high plasma hirudin levels.
The thrombin time shows similar problems to the APTT for monitoring hirudin and shows a non-linear dose response curve and so again is unsuitable for monitoring Hirudin. The Quantitative Thrombin Time [QTT] measures the time to clot formation in a 1:10 diluted patient plasma sample after incubation with fibrinogen and with clotting induced by thrombin. The QTT appears insensitive to heparin, lupus anticoagulants, low fibrinogen, clotting factor concentrations and FDPs but sensitive to hirudin up to 7µg/ml. However, there are limited clinical studies of this test.
The ECT involves the addition of small amounts to Ecarin to plasma and measuring the time to clot formation i.e. the conversion of fibrinogen to fibrin. Meizothrombin is generated by the Ecarin and cleavage at the Arg323 bond is the rate-limiting step in its formation. In the presence of hirudin, meizothrombin is inhibited and so clot formation is prolonged.
The ECT can detect hirudin concentrations as low as 0.05ug/ml and shows a linear response to as high as 5.0ug/ml. The ECT appears relatively independent of variations in the levels of a number of clotting factors including fibrinogen (60-100%) and prothrombin (20-60%).
1. Plasma is diluted with an equal volume of Tris buffer.
2. Measurement of the clotting time starts with the addition of an ecarin solution with a final concentration of 5 Ecarin units/ml.
3. A reference calibration curve is constructed using serial dilutions of a normal plasma pool to which has been added hirudin ranging in concentration from 0-5µg/ml [see Reference 2].
The Single-Step Hirudin Assay: This is a modification of the of Ecarin clotting time in which cuvettes containing lyophilised buffered ecarin are prepared and stored. The cuvettes are prepared prior to use by the addition of distilled water, citrated whole blood is added and the clotting time measured. A pre-stored calibration curve is used to calculate the hirudin concentration in the plasma samples based on its ecarin clotting time.
Modification for High Hirudin Concentrations: In cardiac surgery high levels of hirudin are often achieved due to the low biocompatibility of the oxygenators and the large artificial extracorporeal surfaces. The high hirudin levels needed to achieve efficient anticoagulation for CPB result in long ECTs in the region of 350-450s. To monitor hirudin in this situation, immediately following the collection of the blood sample it is diluted 1:1 with normal plasma and the hirudin is assayed using one of the methods above. This dilution in normal plasma also make the test less sensitive to low fibrinogen and prothrombin levels which are often found during and following CPB.
Chromogenic Assay: The Ecarin Chromogenic Assay [ECA] is similar to the Ecarin Clotting Time but the generation of meizothrombin [and its inhibition by direct thrombin inhibitors] is measured using a specific chromogenic substrate. The ECA shows a linear response in the range from 0.1-0.3µg hirudin/ml plasma. Ecarin chromogenic assays are also available commercially.
In clinical practice, the measurement of hirudin varies between 0.1 -2µg/ml. Plasma levels >2.5 µg/ml represent toxic hirudin levels which are encountered rarely except in cardiac surgery.
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