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Method

How to measure the Thrombogram

The Thrombinoscope Software Program

The Thrombinoscope Software program runs on a Windows PC hooked up to the Thrombinoscope Instrument. With this package it is possible to watch the concentration of thrombin as it develops in clotting (platelet-rich) plasma. All the data collection and reduction is completely automated which makes the method user-friendly and widely applicable. When you are interested to try out the method in practice then please contact us and we will arrange a try-out period in your laboratory and give you, for a limited period of time, a version of the software that has full capabilities.

Add your reagents to the 96-well plate...

...and watch the Thrombin generation on your screen

The Automated Measurement

 

The Subsampling method and the Chromogenic Measurement


The Thrombogram can be assessed in different ways. Traditionally it was measured by determining the concentration of thrombin in subsamples taken at short time intervals from clotting blood or plasma. In 1993 we introduced an automated measurement that was based on the measurement of optical density in PPP. Chromogenic thrombin substrates were used that are converted slowly and do not bind too tightly to thrombin. In this way the clotting mechanism is not disturbed and the velocity of substrate conversion remains proportional to the concentration of thrombin during the course of the thrombin generation. Drawback of the chromogenic method is that it cannot be used to measure thrombin generation in platelet-rich plasma and that the clot formation should be inhibited or defibrinated plasma should be used

The Fluorogenic Measurement


In the laboratories of the university of Maastricht a new method was developed in which the thrombin measurement is carried out in a 96-well plate fluorometer. Here a fluorogenic - in stead of a chromogenic substrate - is used. Since Fluorescence is not heavily influenced by turbidity, the appearance of a clot will not impair the signal so fibrinogen does not need to be removed. Also the presence of platelets does not disturb the measurement. This makes it possible to study the role of platelets in coagulation in large series of samples. All platelet inhibitors tested thus far appear to influence thrombin generation in PRP. Naturally also the plasmatic inhibitors and disorders are reflected in this new assay.

One of the properties of fluorescence is that the amount of signal depends on variables like the aging of filters and lamp, the color and material of the 96-well plate and even the color of the plasma. On top of that, the concentration of fluorophore (we use amino-methyl-coumarin, AMC) is not linear with the amount of signal due to the so-called inner filter effect. Also, during the process of thrombin generation, about 30-40% of the substrate is consumed which decreases the velocity of substrate conversion per concentration of thrombin. This means that not only a correction should be made for the activity of alpha2M-thrombin complex (mentioned above), but also for the inner filter effect and the substrate consumption.

We have developed a patented method using a Thrombin Calibrator that can be added to plasma. This calibrator is now measured in the Thrombinoscope software and compared to the measurement of fluorescence in another well containing the same plasma. With the thrombin calibrator as a reference, we can now calculate the molar concentration of thrombin, during the measurement, as it develops in clotting plasma regardless of the inner filter effect, substrate consumption or ageing of the lamp and filters and donor-to-donor variability in color of plasma.

The result is a facile test in which the user has to fill the wells with reagents usually consisting of a trigger necessary to start coagulation (diluted thromboplastin/Tissue Factor) and plasma. The instrument will dispense a mixture of fluorogenic substrate and calcium so that coagulation begins. After the measurement the program calculates all parameters of the Thrombogram (area under curve, peak height, lag time and more) and expresses the results in nanomolar thrombin in time.

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