A new device for measurement of fibrin clot lysis: application to the Euglobulin Clot Lysis Time

The fibrinolytic system is involved in many physiological and pathophysiological processes. An increase in fibrinolytic activity may induce a bleeding tendency. More commonly, a reduced activity of the system is observed and this may contribute to the pathogenesis of thromboembolic diseases [1, 2]. In earlier clinical studies, an impaired fibrinolytic function had been reported in subjects suffering from venous thrombosis [3]. Newer studies have emphasised the importance of fibrinolytic disturbances in the development of arterial disease and thrombosis [4, 5]. These alterations were especially observed in patients with advanced atherosclerosis, when developing acute-coronary syndrome or diabetes mellitus [6, 7]. The fibrinolytic abnormalities were also observed in hypertriglyceridemic patients before developing atherosclerosis or vascular complications [8]. Most of the tests used to measure the blood fibrinolytic potential are not suitable for routine clinical practices. Analysis of t-PA and PAI-1 considers two isolated components of the fibrinolytic system. Furthermore, these tests have to be performed in series and so cannot be done very frequently in laboratory routine for logistic and economic reasons. The Dilute Blood Clot Lysis Time is a very long and subjective procedure requiring a continuous monitoring by an observer who has to determine when the clot is lysed. The Euglobulin Clot Lysis Time (ECLT) is the most commonly used test. It seems to represent the interaction between the activity of t-PA and PAI-1 [11] and allows to measure both baseline and stimulated fibrin dissolution. Although this test is inexpensive and adapted to single tests system, it is very difficult to promote in clinical practice, since it is long, and require an observer to record the clot lysis.

We have designed a new semi-automatic method for clot lysis measurement that includes several important improvements which are: 1) the device is operator independent during the analysis of the fibrinolysis process and for the determination of the lysis time, 2) allows the analysis of samples with a broad range of lysis time, 3) allows the analysis of individual samples and is easily integrated in the laboratory routine. To answer to these points, our new system has been completely computerised. ECLT has been chosen as first application. We have compared the efficiency of ECLT with the new device to the classical manual method and to a new test from Stago, the Global Fibrinolytic Capacity (GFC). In addition, the test was also performed on high blood triglycerides level, to test the optical sensitivity and robustness of our device.

Our aim was to create a device which allowed to reintroduce the fibrinolysis measurement in routine practice without disturbing the laboratory workflow, in this work we chose ECLT. An optical computerised system for the follow-up of fibrinolysis process and for determination of lysis time made the test autonomous and allowed further developments. With our device, we obtained a very tight correlation with the manual method, but this one has been made in condition unachievable in routine. The response to venous occlusion were identical for manual and semi-automatic method.

We have obtained a significant difference between normal values in men and premenopausal women in accordance to the notion that women have a better fibrinolytic capacity than men [15].

Another procedure, allowing quantitative ECLT assessment with a microtiter plate reader, was reported by Urano et al [11]. It provided reliable and reproducible data, with the lysis time determined as the half lysis point obtained by calculation of the midpoint between maximum and minimum points of turbidity. However, the turbidity of the clot was only measured every 30 minutes by the microtiter plate reader. We used a higher sampling rate of the recorder, i.e. every one minute. Our mathematical analysis theoretically allows a better accuracy since it is based on much more discrete measurements than in the system of Urano using only an unique half lysis point.

Moreover, our device allows to analyse up to 8 samples individually and to measure the lysis time over a broad range (5 minutes to 9999 minutes) strictly stabilized at 37°C. The mathematical analysis determines not only the critical points (A, B, C) of the lysis curve, but will also enable to explore the kinetics of fibrin clot degradation in the future.

Finally, this device is able to determine the lysis time in hyperlipemic condition. The GFC from Stago can also be applied on single samples. This test evaluates the fibrinolytic process by the generation of D-Dimer from an external and standardized fibrin clot. It is rather expensive due to the reagent itself and the cost of DD evaluation. At first sight, we thought that the fibrinogen level had no effect on GFC and that this test could result in a different information than ECLT. But, both tests are surprisingly, equally sensitive to patient's fibrinogen level. Fibrinogen can indirectly or directly interfere with fibrinolysis process. The first mechanism may be common to both tests, the fibrinogen level can represent chronic inflammation which may impaire fibrinolytic balance [16].

The second mechanism (direct effect) is probably different for ECLT than for GFC. For ECLT, the density of fibrin clot is in relation with fibrinogen level and this density may also modulates lysis time [17]. For GFC, a constant amount of t-PA is added in plasma sample which is placed at the surface of a standardised fibrin clot (the reaction is stopped after one hour). A previous study [18] reported that t-PA incubated with plasma in presence of a fibrin clot induced fibrinogen proteolysis. The authors concluded, that the products of t-PA-induced degradation of cross-linked fibrin could potentiate t-PA-mediated fibrinogenolysis by providing a surface for t-PA and plasminogen binding thereby promoting plasmin generation. This plasmin is a more important mediator of fibrinogenolysis than plasmin generated on the clot surface. We suppose that, a part of plasma plasminogen is less available for the standardised fibrin clot degradation and that this phenomenon can induce a decrease of D-Dimer generated during the test.

The GFC shows a weak sensitivity in hypofibrinolytic patients with no possibility to distinguish between two different samples or to measure a first slight response to a treatment. Alternative protocols should be designed to allow an evaluation of these samples. Several tests on plasma were achieved on samples providing from venous occlusion and hepatic transplantation (data not shown). The device determine easily the lysis time on plasma. However, in basal condition fibrinolysis is too slow. Different preparations of reagents are in development, to reduce lysis time, for the new applications with our device. The new apparatus is an important tool for studying fibrinolysis in-vitro, moreover, it can reintroduce lysis time in routine for a better approach of hypofibrinolysis.

It has allowed us to achieve preliminary studies on the effect of lipids level on ECLT in men and women [19] and the relations between the classical cardiovascular risk factors and ECLT [20].

In conclusion, we observed a good correlation between the measurement of lysis time by the manual method and the new device. This new evaluation of the ECLT is more objective. The results are highly reproducible and the test easy to perform. The reagents and disposable are not expensive (micro-cuvets, acetic acid, Owren solution and thrombin). At the moment, it is integrated in our laboratory routine practice for extended evaluation.

This ECLT test should be reintegrated in clinical routine, and could be conveniently added to the panel of estimates of biological risk factors for atherothrombosis. Moreover, this device is not limited to ECLT, it could be used with different sample preparations.