Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
2000-01-24
2002-08-13
Gitomer, Ralph (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C435S004000, C436S069000
Reexamination Certificate
active
06432657
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention mainly relates to a method to determine coagulation parameters such as prothrombin time, activated partial thromboplastin time and the like in a sample containing fibrinogen, for example a plasma sample, as well as to a kit to conduct the method. As one of the features of the invention is that since the coagulation parameters can be measured without making a sample completely clotted, an automated analyzer can be easily applied to the operation.
2. Description of the Related Art
Currently, most of the methods for measuring (or determining) the coagulation parameters in a sample containing fibrinogen such as a plasma sample, are based on addition of a blood coagulation reagent to the sample in a reaction vessel, incubation thereof, and optical or physical detection of the resulting fibrin clot. Specifically, the coagulation parameters are measured by monitoring the degree of an increase in turbidity or viscosity of a sample accompanied by the production of stable fibrin from the fibrin clotting process illustrated hereinafter.
Accordingly, fibrin clot which occurs when a blood coagulation reagent is added to a fibrin-containing sample is roughly composed of (1) a process in which a coagulation cascade reaction starts to produce thrombin, (2) a process in which the produced thrombin react as a catalyst upon fibrinogen existing in the sample to make fibrinopeptide A freed from the A&agr; chain of fibrinogen and fibrinopeptide B freed from the B&bgr; chain, and to produce a fibrin monomer on which E region the polymerization site “A” and the polymerization site “B” are bared, (3) a process in which the produced fibrin monomers bind non-covalently each other right after another through an interaction between the polymerizing site “A” existing in the E region and the polymerizing site “a” existing in the D region, and (4) a process in which upon the resulting fibrin polymers factor XIII activated by thrombin (activated factor XIII) reacts and the adjacent D regions form cross-links (that is covalent bonds) through cross-linking reaction resulting in stable fibrins, and the conventional methods measure the coagulation parameters through monitoring the degree of the increase in turbidity or viscosity of the sample accompanied by progress of the above-mentioned process (4).
Here, as the types of the coagulation parameters measurable in these methods, blood coagulation times such as prothrombin time (PT) to inspect whether the extrinsic pathway of coagulation is normal or abnormal, activated partial thromboplastin time (APTT) to inspect whether the intrinsic pathway of coagulation is normal or abnormal; thrombotest and hepaplastin test to investigate whether vitamin K-dependent coagulation factors are normal or abnormal; the % activities of coagulation inhibitors such as Protein C, Protein S and the like; the plasma contents of each coagulation factor relating to the coagulation cascade reaction such as factor II, V, VII, VIII, IX, X, XI, XII and the like; and plasma levels of fibrinogen and the like can be mentioned.
In the above-mentioned conventional methods for measuring the coagulation parameters, however, because of the need to produce stable fibrin due to the principle for the measurements, it has been substantially impossible to use automated analyzers for the measurements. Accordingly, because the resulting stable fibrin will clot and firmly stick to a reaction vessel (such as a cuvette, cup and the like), and then it can not be removed without manual operations such as removal using a spatula, the reaction vessel can be used only once for the measurements of the coagulation parameters. Because of this reason, the reaction vessel after the measurement has to be disposed and thus the methods cannot be applicable to automated analyzers which repeatedly use the reaction vessels. Accordingly, under the current situation, only the measurements of the coagulation parameters are measured using an exclusive devise for blood coagulants independently of many other test items (the test items for which biochemical reagents and immunological reagents are used) which can be all measured in one automated analyzer.
Although some methods to solve the above-mentioned problems have been reported, none of them has been put to practical use. For example, Japanese Unexamined Patent Publication No. 7-255497 discloses a method wherein the coagulation parameters are measured when fibrin clotting process is forced to stay within the phase of fibrin polymer formation (where the fibrin monomers are precipitated as fibrin net) by adding an inhibitor of factor XIII to the reaction system, and after the measurements, the fibrin polymers are dissolved by addition of a solution to remove the affinity among the fibrin molecules. This method also indicates that used reaction vessels can be reused. As any of the inhibitors against factor XIII used in this methods is, however, a dangerous drug, safety must come into question. Moreover, whereas the said method requires the addition of a solution to remove the affinity among the fibrin molecules aiming at dissolving fibrin polymers, those specifically exemplified as the said solutions are strong acids such as hydrochloric acid which may corrode or damage the reaction vessel (such as a cell) and the measuring devise itself. In addition, as these solutions are added after completion of coagulation, a new line for addition of the solution is necessary to be established in the measuring devise, indicating a problem that an automated analyzer which has already used commonly can not be used as it is.
Still, no description is presented in the above-mentioned publication about reproducibility of the measured data, consistency with the conventional methods for measuring the coagulation parameters using multiple samples and the like, leaving credibility on these issues unclear.
As mentioned hereinbefore, to the best of the inventors' knowledge, no method for measuring the coagulation parameters, which is simple and applicable to existing automated analyzers has been described. Thus, the invention aims at development of a method for measuring the coagulation parameters, which is credible and applicable to existing automated analyzers.
SUMMARY OF THE INVENTION
Guided by an idea that the above-mentioned purpose can be achieved by measuring the coagulation parameters without generating fibrin clot (or stable fibrin) and then fibrin net in a reaction vessel, the inventors conducted a diligent investigation on a method which brings about a change in a physical value of the sample corresponding to the coagulation parameters without generating fibrin clot and fibrin net. As a result, it was found that by suppressing and inhibiting the formation of non-covalent bond among fibrin monomers which is catalyzed and produced by thrombin, and then avoiding the formation of fibrin clot and further fibrin net, a change in a physical value of the sample before the formation of fibrin net (i.e., fibrin polymers are deposited out) shows an extremely close correlation to any type of the coagulation parameters.
Accordingly, the invention enables determination of the coagulation parameters in a fluid sample before the said sample to be tested completes clotting by utilizing the above-mentioned correlation.
Such an invention is a method for determining the coagulation parameters in a clotting system comprising fibrin clotting process accompanied by limited degradation of fibrinogen by thrombin, which comprises of;
A. a process to contact a fibrinogen-containing fluid sample to be tested with a blood coagulation reagent at the presence of a material which inhibits the formation of fibrin polymers from fibrin monomers in the aforementioned fibrin clotting process,
B. a process to detect the change in a physical value of the said sample which changes is triggered by such a contact, and
C. a process to evaluate the change in the physical amount detected as an indicator presenting a coagulation parameter.
Furthermore, this invention
Kikuchi Masayoshi
Watanabe Shin
Yoshimura Yoshimichi
Chaudhry Mahreen
Gitomer Ralph
Tokuyama Corporation
Wenderoth , Lind & Ponack, L.L.P.
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