Population data
The normal values in non-stimulated condition were obtained from 25 women and 21 men. Men were of 49 years old (range, 32–54) and women 44 years old (range, 31–48) (p = 0.3). Volunteers were free from any clinical signs of peripheral arterial disease, diabetes mellitus, arterial hypertension, history of cardiovascular disease, inflammatory syndrome and smoking habit. They were also free of medication and women were in premenopausal status. For correlation with the manual method, blood samples from others 11 fasting volunteers from laboratory were obtained before and after a 10 minutes venous occlusion. The relation with the Global Fibrinolytic Capacity (GFC) was established on 42 samples obtained from patients explored for thromboembolic disorders. Among these, 25 gave another sample after a 10 minutes venous occlusion. The stimulation conditions were also evaluated on patients, taken 0.5 hour, 1 or 2 hours after desmopressin infusion (therapeutic challenge) in mild haemophilia A and von Willebrand patients.
Blood sampling
For the normal values, blood samples from antecubital vein were obtained between 08:00 and 09:30 a.m. Samples were harvested in 0.13 M-citrated vacuum tubes (Venoject® Terumo) and immediately put into melting ice. After centrifugation at 4000 g for 10 min at 4°C, the plasma samples were stored at -80°C until analysis. The tests were performed either immediately or on frozen/thawed samples (-80°C). Fibrinogen level was tested on STA® apparatus according to manufacturer's instructions.
Euglobulin fraction preparation
300 μl of acetic acid (0.25%) and 3.6 ml of desionized water were added to 400 μl plasma (final pH ≅ 5.9). The sample was then put into melting ice during 20 min and centrifuged at 4000 g for 10 min at 4°C. The supernatant was discarded and the pellet was resuspended in 400 μl of Owren-Koller buffer (DIAGNOSTICA STAGO®, France). The clot formation started when 100 μl of thrombin (1.75 U/ml, DIAGNOSTICA STAGO®, France) were added.
ECLT measurement: conventional manual method
The euglobulin activated fraction was put at 37°C in a thermostatic bath and the clot was carefully examined every 5 minutes by the operator who was continously present during the measurement. Lysis was considered complete when the clot was no more apparent (end time point expressed in min).
Semi-automatic ECLT measurement
The procedure is very easy, the sample (euglobulin fraction, plasma or others) is introduced in a microcuvette and put in the apparatus, in a well. Then, the operator follows three operations to start the tests: 1- identification of sample, 2- add thrombin, 3- push on set button to start the recording of fibrinolysis process.
The new device composition was designed as Figure 1:
– a thermostatic bloc (37°c) with eight photometric measure channels.
– a programmable language control (PLC).
– a computer and a software for the mathematical analysis of the fibrinolysis process.
Thermostatic bloc
the bloc is made of pure aluminium (360 × 30 × 100 mm) and is warmed by two resistances of 20 W. It is designed to insert spectrophotometric micro-cuvets (10 × 4 × 45 mm Sarstedt®) into 8 wells. Each well includes one emitter (diode: SFH 409) and one receptor (phototransistor: SFH309FA), both operating at 890 nm. When the clot is formed, the phototransistor signal decreases reflecting a Tyndall effect [12].
Programmable Language Control
ED&A V10, Antwerpen, Belgium. The sensors are connected to the PLC which transforms the analogic data (0–10 V) into digital data (12 bits). The data are transmitted to the computer by RS232.
Data analysis
The computer records every one minute the data from each channel. The software (developed with Windev® 4.1, France) generates the graph of the fibrinolytic process. At the end, the curve graph is analysed with a mathematical algorithm. The first and second derivatives are computed by a convolution matrix13. These calculations aim at determining the peak time to clot formation (A point, first derivative), the peak time to clot lysis (B point, first derivative), and the end of the complete fibrinolysis process (C point, when the first and the second derivatives are under the background ≅ 0). Later, other parameters should also be considered. The design of a lysis curve is exemplified in Figure 2. The x-axis/y-axis respectively represent time and evolution of the signal sensor. Mathematical analysis or complete procedure can be started individually for each sample and at every moment without disturbing the measurements of other samples. The final assembly was performed by EREM, (Marcinelle, Belgium) and the system is distributed since 2001 by BIOK, (Montigny-Le-Tilleul, Belgium).
The Global Fibrinolytic Capacity (Stago, France)
This test estimates the fibrinolytic activity in plasma through the measurement of generated D-Dimer from a standardized fibrin amount in presence of a constant amount of exogenous t-PA and by contact phase activators [14]. The test was performed according to manufacturer's instructions and the results were expressed as generated DD (μg/ml).
Test of hypertriglyceridemic status
To assess the blood triglycerides effect on the optical property of the euglobulin fraction, two samples were obtained from one hyperlipemic volunteer in preprandial and 2 hours postprandial states. The ECLT was examined on both samples with a photographic follow up. The total cholesterol and the triglycerides were determined by routine laboratory methods, on a Synchron LX20 (Beckman®).
Statistical analysis
SigmaStat® Jandle Scientific software was used. Non parametric statistical analysis with Spearman test for the correlations and Mann-Whitney test for the comparison between men and women were used. The relation between the GFC and the semi-automatic method was described by an exponential equation. Data are presented as median values and ranges (25%–75%). A p value < 0.05 was considered as statistically significant.