Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2002-09-27
2004-06-15
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C436S069000, C436S533000
Reexamination Certificate
active
06750032
ABSTRACT:
The invention relates to a method and to a diagnostic agent for detecting hemostasis disturbances, wherein, as a consequence of blood platelet aggregation, clot formation and/or clot dissolution, substances are brought to a distance from each other which permits or prevents an interaction, in particular an energy transfer, between the substances, and the extent of the interaction is measured.
The bloodstream supplies the organs with nutrients, and provides for the disposal of metabolic products, by way of the vascular system. The maintenance of an open vascular system, which is nevertheless sealed off against the environment, is therefore of vital importance. This is made possible by the interaction of the opposing systems of blood coagulation and fibrinolysis—which are in an equilibrium, termed hemostasis, with respect to each other. In extreme cases, a disturbance of these systems is manifested clinically either in thromboses, i.e. unintentional occlusions of the vascular system, or in a hemorrhagic diathesis, i.e. bleeding. Both phenomena can lead to death by way of organ failure. They represent one of the main causes of mortality in the western world. An important role is therefore attached in clinical diagnosis to detecting an acquired or inherited hemostasis disturbance.
The processes of hemostasis are based on the interaction of blood vessels (contraction and dilatation), cells (the endothelium lining the blood vessels and/or thrombocytes which are floating in the blood vessels) and humoral factors. A distinction is made between primary and secondary coagulation in accordance with the physiological sequence of the processes. The cellular components dominate in primary coagulation and this coagulation concludes with the formation of thrombocyte aggregates (primary clot). The humoral factors dominate in secondary coagulation, which is normally initiated by the cellular components. These humoral factors are proteins, which are essentially differentiated, in accordance with their function, into enzymes, cofactors and supporting proteins. When coagulation is activated, active or activated enzymes proteolytically convert proenzymes, by way of a cascade-like system, into their active form. This activity can be increased by means of cofactors which are for the most part themselves proteolytically activated. As the last step, the supporting protein fibrinogen, which is soluble in its precursor form, is converted by the coagulation enzyme thrombin into its insoluble resultant product, i.e. fibrin. The fibrin clot (secondary clot) is produced by the aggregation and enzymic crosslinking of the fibrin. Primary and secondary coagulation promote wound healing. Under pathological circumstances, however, the bloodstream becomes unintentionally blocked, an event which is clinically described as thrombosis. The clots are dissolved by the fibrinolysis system, which leads, by way of a similar succession of activating enzymes, to activation of the protease plasmin. Under the pathological circumstances of hyperfibrinolysis, plasmin nonspecifically destroys fibrinogen which has still not clotted and thereby disrupts the integrity of the capillary system, resulting in bleedings.
Diagnostic methods, which are differentiated into so-called
classical methods,
clinical chemistry methods, and
immunochemical methods,
are used to investigate the above-described processes for the purpose of detecting possible disturbances of hemostasis.
The diagnostic methods which are termed classical methods are based on detecting the formation of a clot. Those methods predominate which activate the activation cascades of coagulation and/or of fibrinolysis at one point and measure the time until a clot has formed or dissolved. Changes in these coagulation or fibrinolysis times as compared with a normal sample enable conclusions to be drawn with regard to pathological changes which have taken place in the constituent section which has been activated, i.e. the section of the reaction cascades up to the formation or dissolution of a fibrin aggregate. In the classical methods, the clot is normally detected by means of mechanical or optical detection methods.
Mechanical detection uses the increased viscosity of the coagulating sample or the formation of fibrin threads. For example, a sphere or a stirring flea is placed in the bottom of a reaction vessel and coagulation is induced in the blood sample which is added to the vessel. The sphere or the stirring flea or the vessel rotate and the force required for this movement is measured. If the viscosity of the solution increases due to platelet aggregation or clot formation, this is measured as an increased resistance and, from a particular value onwards, assessed as being the onset of coagulation. In another method (in accordance with Schnitger & Gross), a hook-shaped electrode is dipped into the sample and withdrawn from it at regular intervals. In association with this, the supply of current to an electrode which is lying in the sample is switched on and off in a corresponding manner. If a fibrin thread is formed, the mobile electrode becomes entangled and the flow of current is maintained. This is interpreted as the onset of coagulation. Conversely, when fibrinolysis is being diagnosed, the dissolution of the clot is detected in a corresponding manner by a decrease in the viscosity or the dissolution of the fibrin.
In the classical methods, mechanical detection has the advantage that it registers the physiologically important property of clot formation, namely the mechanically stable closure of a wound. On the other hand, this technique requires special equipment which is only suitable for these special hemostasis tests.
Optical detection is understood to mean turbidimetric determination of the change in the turbidity of the solution when a clot is formed. The turbidity signal associated with a fibrin clot can be amplified by means of methods which are known to the skilled person, for example by means of increasing the optical density of the sample, for example by adding particles, or by means of augmenting the denaturation of the fibrin by adding salts or ions, for example metal ions such as manganese, iron or calcium ions. In the classical methods, optical detection is not understood to mean the conversion of chromogenic substrates, which can likewise be measured photooptically, since this records an enzyme activity and not the aggregation of thrombocytes or conversion of the natural supporting protein fibrinogen.
EP 0 039 885 reports that hydrophobic latex particles can be used for detecting fibrin monomers, i.e. fibrin which has still not polymerized. However, such simple latex particles are not suitable for detecting a clot since the agglutination of latex particles causes increased light scattering which cannot be distinguished from the light scattering which a fibrin clot itself produces.
An advantage of optical detection is the possibility of carrying out the measurements on current clinical chemistry automated analytical equipment. However, since optical detection is based on measuring turbidity, turbidities in samples, e.g. in the case of hyperlipemic samples, can, in contrast to mechanical detection, interfere with the determination. In extreme cases, for example in the case of whole blood, no optical detection is possible at all, due to the optical density. This is an important disadvantage of these optical methods.
In addition to the above-described conventional classical diagnostic methods, the clinical chemistry methods are also customarily used in hemostasis diagnosis. These methods use the conversion of specific, chromogenic substrates, on their own or in combination with enzymes and/or intermediates (enzymically coupled test) to measure the activity of individual enzymes. These determinations are independent of the formation of a thrombocyte aggregate or of a fibrin clot. A disadvantage in this context is, however, that the physiologically relevant reactions, namely disturbances in the formation of a thrombocyte aggregate or a fibrin clot, or their dis
Kraus Michael
Schelp Carsten
Wiegand Andreas
Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Weber Jon P.
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