Method for determining the spatiotemporal distribution of activity of a proteolytic enzyme in a heterogeneous system (variations), a device for realizing same and a method for diagnosing the defects in the hemostatic system on the basis of a change in the spatiotemporal distribution of activity of a proteolytic enzyme in a heterogeneous system

A method for determining the spatiotemporal distribution of activity of a proteolytic enzyme in a heterogeneous system, comprising the steps of:

• providing an in-vitro system that includes a sample of a test medium, wherein the test medium is selected from the group consisting of blood plasma, whole blood, water, lymph, colloidal solution, crystalloid solution and a gel, and a proteolytic enzyme or its zymogen, wherein the proteolytic enzyme or its zymogen is distributed in the sample of the test medium;
• combining a chromogenous substrate with the in-vitro system, wherein the chromogenous substrate is cleaved by the proteolytic enzyme, and a chromogenic mark is released upon cleavage of the chromogenous substrate;
• illuminating the sample of the test medium by light having a wavelength corresponding to substantial absorption thereof by the chromogenic mark;
• recording the spatial distribution of light absorption of the chromogenic mark in the sample of the test medium, and obtaining a set of distributions of the light absorption points;
• calculating the spatiotemporal distribution of the proteolytic enzyme activity by using the set of distributions of the light absorption and solving the inverse “reaction-diffusion-convection” problem,
• wherein the step of calculating includes correcting the spatiotemporal distribution by using at least one numerical algorithm for reducing noise and distortion of the chromogenic signal.

A method for determining the spatiotemporal distribution of activity of a proteolytic enzyme in a heterogeneous system, comprising the steps of:

• providing an in-vitro system that includes a sample of a test medium, wherein the test medium is selected from the group consisting of blood plasma, whole blood, water, lymph, colloidal solution, crystalloid solution and a gel, and a proteolytic enzyme or its zymogen, wherein the proteolytic enzyme or its zymogen is distributed in the sample of the test medium;
• combining a luminescent substrate with the in-vitro system, wherein the luminescent substrate is cleaved by the proteolytic enzyme and a luminescent mark is released upon cleavage of the luminescent substrate;
• recording the spatial distribution of a luminescence signal of the luminescent mark in the sample, and obtaining a set of distributions of the luminescent signal;
• calculating the spatiotemporal distribution of the proteolytic enzyme activity using the set of distributions of the luminescent signal and solving the inverse “reaction-diffusion-convection” problem,
• wherein the step of calculating includes correcting the spatiotemporal distribution by using at least one numerical algorithm for reducing noise and distortion of the luminescent signal.

A device for determining the spatiotemporal distribution of activity of a proteolytic enzyme in a heterogeneous system, wherein the device consists of:

• an in-vitro system that includes a cuvette to place the sample of a test medium, wherein the test medium is selected from the group consisting of blood plasma, whole blood, water, lymph, colloidal solution, crystalloid solution and a gel, and a proteolytic enzyme or its zymogen, wherein the proteolytic enzyme or its zymogen is distributed in the sample of the test medium, and a substrate, wherein the substrate is selected from the group consisting of a fluorogenic substrate, a chromogenic substrate, and a luminescent substrate;
• means to illuminate the sample of the test medium in determined times;
• means to record the spatial distribution of a signal of a mark released upon cleavage of the substrate by the proteolytic enzyme;
• means to control illuminating means and recording means;
• means to provide constant pressure within the volume of the sample of the test medium;
• means to illuminate the sample of the test medium using dark-field microscopy.

A method for diagnosis of haemostatic system disorders in a subject based on the change in the spatiotemporal distribution of activity of a proteolytic enzyme in a heterogeneous system in-vitro, comprising the steps of:

• providing a test sample obtained from the subject that includes blood components selected from the group consisting of:
• whole blood, platelet-free blood plasma, platelet-poor blood plasma, platelet-rich blood plasma, blood with addition of anticoagulant, and blood plasma with addition of anticoagulant;
• providing conditions for formation of a proteolytic enzyme in the test sample by performing at least one operation selected from the group consisting of:
• contacting the test sample with the blood coagulation activator immobilized on a surface;
• adding a substrate to be cleaved by the proteolytic enzyme;
• adding a calcium salt;
• adding an inhibitor of the contact activation of coagulation;
• recording at determined moments of time the spatial distribution of the signal of a mark released upon cleavage of the substrate by the proteolytic enzyme, and obtaining a set of spatial distributions of the signal of the mark;
• calculating the spatiotemporal distribution of the proteolytic enzyme activity by using the set of spatial distributions and solving the inverse “reaction-diffusion-convection” problem, wherein the proteolytic enzyme is a coagulation factor;
• wherein the step of calculating includes correcting the spatiotemporal distribution by using at least one numerical algorithm for noise and distortion of the mark signal;
• assessing the state of hemostasis in the subject based on the spatiotemporal distribution of the proteolytic enzyme activity by comparing the spatiotemporal distribution to a proper control.