JPH06273422A - Method for determining quantity of coagulation factor of blood - Google Patents

Abstract

PURPOSE:To make it possible to perform accurate, quick measurement by obtaining the correlation between the coagulation time and the coagulation factor quantity in blood, which are obtained by measuring the temperature change of a heating sensor. CONSTITUTION:A heating sensor, which can has heating action and can measure its own temperature, is brought into contact with blood thermally. The coagulation time is measured from the temperature change of the heating sensor during the time from the input of coagulating agent to the coagulation. The quantity of the coagulation factor in the blood is determined base on the correlation between the coagulation time and the coagulation factor quantity. The correlation between the coagulation time and the coagulation factor quantity is measured by using the heating sensor, which has the heating action and can measure its own temperature and the coagulating time corresponding to diluent by changing the concentration of the blood. The correlation of both values is obtained by forming the calibration curve indicating the correlation between the coagulating time and the coagulation factor quantity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying blood coagulation factors, which enables accurate and rapid measurement of blood coagulation factors.

[0002]

2. Description of the Related Art Quantification of blood coagulation factors is important as screening of blood coagulation system and emergency medical information, and has great clinical significance. Currently, the blood coagulation factor is 1
Three types are known. The blood coagulation system is roughly divided into an endogenous system having a reaction initiation point of factor X2 or factor V3 and an extrinsic system having a tissue factor and factor V2 as a reaction initiation point. At the final stage of the reaction, prothrombin is activated by activation of the coagulation system. , Activated by thrombin, acts on fibrinogen in plasma to form fibrin, and is completed by conversion to insoluble fibrin by the action of factor X3. As a technical document relating to blood coagulation, "Research on Clinical Laboratory Methods", Kinbara Shuppan Co., Ltd., pages VI-72 to VI-77 describes the relationship between coagulation factors and diseases. "Chart Clinical Laboratory Diagnosis", pages 105-126, published by Chugai Medical Co., Ltd.
It is described in detail in the supplementary issue of 9 years "Extensive blood / urine chemistry test, immunological test (first volume)" issued by Nihon Rinjo Co., Ltd., pp. 807-824. By the way, the quantitative measurement of each coagulation factor in blood has been conventionally carried out by an optical measuring method or an antibody reaction, but there are problems such as repetitive accuracy and long measurement time.

The following are examples of prior art relating to blood coagulation. ． Japanese Unexamined Patent Publication No. 54-23596 "Blood Fibrinogen Quantification Method" is one of optical measurement methods for quantifying fibrinogen from the correlation between the change in absorbance at a specific wavelength due to a change in coagulation of plasma and the concentration of fibrinogen. Is. ． Japanese Unexamined Patent Publication No. 1-147367 "Coagulation Factor Assay Method and Assay Reagent" uses a monoclonal antibody to quantify the coagulation factor, and reacts with an insoluble carrier reagent sensitized with the monoclonal antibody against the coagulation factor. The amount of aggregated particles is compared with the amount of aggregated particles, and the coagulation factor is quantified from the correlation between the ratio and the concentration of fibrin degradation products.The amount of reagent is adjusted according to the target coagulation factor. is there. ． Japanese Patent Application Laid-Open No. 1-227062 “Measurement Method of Viscosity Change of Blood etc.” uses a sensor composed of a heat absorbing body or a heat generating body to measure the viscosity change from the measured temperature, etc. It is a way to know the time.

[0004]

By the way, the conventional measurement of coagulation factors is time-consuming and cannot be applied to emergency medical care, requires special techniques, instruments, reagents, etc., and the unit cost of measurement is expensive and artificial. There is a problem that reproducibility is poor and reliability is poor in measurement methods that involve physical judgment. Further, the invention of the above is an example of an optical measurement method, but the absorbance is measured using a spectrophotometer. There are measurement restrictions such as that the sample container must be transparent and that whole blood cannot be measured.
In addition, it was necessary to prepare various measurement environments essential for optical measurement. The above-mentioned invention is an example of utilizing an antibody, but as a method of utilizing an antibody, there is a method of performing quantification using a colorimetric method in addition to this invention. In the method using such an antibody, a special antibody is first required regardless of which assay method is finally used, and the problem is that the antibody itself is expensive. This will directly affect the measurement cost, such as when repeating the measurement in accordance with clinical standards. Further, in the measuring method using an antibody, the pretreatment of the sample and the measuring method are complicated, the measuring time cannot be drastically shortened, and a highly skilled person in charge of measuring is required. A further invention is a viscometric method for accurately measuring the coagulation time using a sensor that is in thermal contact with blood and the like, but in the present invention, clinically effective quantification of coagulation factors is disclosed. Not not.

[0005]

The present invention uses a thin wire heating method for thermally measuring the physical properties of a fluid, has a low measurement unit price, does not involve human judgment, and is a quick and accurate quantitative determination of blood coagulation factors. It is intended to provide the law. The configuration is a heat generation sensor that has a heat generation function and can measure its own temperature by making thermal contact with blood, serum, or plasma or a diluted solution thereof, and from adding the coagulant to coagulation. The coagulation time is measured from the temperature change.
Then, the amount of coagulation factor in the blood, serum or plasma is quantified from the correlation between the time and the amount of coagulation factor. Correlation between clotting time and amount of clotting factor is
Correlation between coagulation time and the amount of coagulation factor by measuring the coagulation time corresponding to the dilution series by using a fever sensor capable of measuring the temperature of itself by changing the concentration of the diluted solution of serum or plasma The correlation can be obtained by creating a calibration curve indicating It is preferable to quantify the coagulation factor using this correlation. By the way, a method of measuring the temperature of a heat generating sensor by bringing the heat generating sensor into thermal contact with blood, serum, or plasma or a diluted solution thereof is called a fine wire heating method. Are roughly classified into the unsteady method and the stationary method. The non-steady state method is an increasing change range (non-steady state) of the temperature of the heat generating sensor or the temperature difference between the temperature of the heat generating sensor and the temperature of the fluid immediately after the heat generation of the heat generating sensor that is in thermal contact with the fluid starts.
Is a method of measuring the thermal conductivity of a fluid, etc., and the steady-state method is a method in which the temperature of the heat-generating sensor or the temperature difference between the temperature and the fluid becomes constant over time after an unsteady state has passed. It is a method of measuring the state of a fluid by utilizing it. When the physical properties of the fluid change in this steady state, the temperature of the exothermic sensor or the temperature difference between the temperature and the fluid changes to a different temperature and stabilizes again. This is because the heat transfer coefficient on the surface of the heat generation sensor changes due to changes in the physical properties of the fluid. It is the basis of the present invention that the change in physical properties of a fluid, in particular, the change in viscosity can be accurately and promptly measured by a thermal method by utilizing such a convection heat transfer phenomenon. The present invention further clarifies the clinical meaning of these measurement results in the form of quantification of coagulation factors. In other words, clarify the relationship between the measurement time of blood coagulation time using the thin wire heating method and the coagulation factor specified by the type of coagulant used for this measurement, and use this to quantify the coagulation factor. To do.

[0006]

[Function] The blood is coagulated after the coagulant is added by using a method in which a heat generating sensor that has an exothermic effect and can measure its own temperature is brought into thermal contact with blood, serum, plasma or a diluted solution thereof. The coagulation time is measured from the temperature change of the heat generation sensor until the time. Then, the amount of coagulation factor in the blood, serum or plasma is quantified from the correlation between the time and the amount of coagulation factor measured in advance. The correlation between the coagulation time and the amount of coagulation factor depends on the concentration of the dilute solution of blood, serum or plasma, the coagulation time corresponding to the dilution series has an exothermic action, and a fever sensor capable of measuring its own temperature. The correlation can be obtained by making a measurement by using the calibration curve showing the correlation between the coagulation time and the amount of the coagulation factor.

[0007]

【Example】

(Example 1) The thrombin time (TT time) in a dilution series of normal human plasma (human normal plasma for measuring blood coagulation ability, manufactured by Organon Technika Corp.) was measured, and the TT time was suggested by the TT time and the coagulation factor. It shows the correlation with the fibrinogen concentration. As a dilution series of Plate Poor Plasma (PPP), 10%, 5%, 2.5% of physiological saline is used in accordance with the standard method based on Nippon Rinko.
%, 1.25% diluted anticoagulant added sample was used.
1A is a heat generation sensor (diameter 0.6 mm, length 4 m)
The measurement value of the heat generation value of m) was 0.02 Watt, which is the measurement result of the coagulation time when a sufficiently large amount of thrombin was added as a coagulant. The change is continuously measured from the time when the coagulant is added. As shown in the figure, since the heat generation of the heat generation sensor is started at the same time as the addition of the coagulant, the temperature of the sensor rises at the beginning of the measurement and shows an unsteady state during the rising process, and then a steady state that stabilizes at a constant temperature. Is shown. After that, when the change in coagulation rapidly appears, the change in the sensor temperature rises again. This is because the change in solidification was observed as a change in viscosity, and the time at this time is the solidification time. B of FIG. 1 shows the time change rate of the time-temperature difference curve of A of FIG. 1, and the table below shows the measurement results of the solidification time defined by the inflection point of the time-temperature difference curve. It is shown.

[0008]

[Table 1] From this table, it can be seen that the reproducibility of the measured values by the method of the present invention is practically sufficient.

FIG. 2 is a calibration curve showing the correlation between the TT time and the plasma concentration (VNC) corresponding to the amount of coagulation factor prepared based on this experiment. The coagulation factor corresponding to TT time is fibrinogen.

(Embodiment 2) Under the same conditions as in Embodiment 1, 10
FIG. 4 shows the results of measuring the activated partial thromboplastin time (APTT time) using diluted whole blood of% concentration.
Similarly to FIG. 1, A and B of FIG. 1 are continuous measurements of the temporal change in the temperature of the exothermic sensor in this experiment from the time of solidification. This is because a change in coagulation was detected as a change in viscosity of diluted whole blood. If the coagulation time was defined as in Example 1, the coagulation time was calculated to be 249.3 seconds in this example. FIG. 3 is a calibration curve showing the correlation between the APTT time and the plasma concentration corresponding to the amount of coagulation factor regarding plasma obtained by diluting to 5%, 2.5%, and 1.25% based on this measurement. This figure was obtained by polynomial approximation by the least desperation method. In the figure, Actin, Platelin Plus A
The ctivator and aActivated Thrombofax are reagents for measuring APTT, and different coagulation times were observed in the same sample depending on the type of reagent, and it was found that the disparity between reagents increased as the concentration of the sample decreased. However, in this case, the activity is measured in the state that four coagulation factors related to the APTT time of the endogenous system are included, and in the case of abnormality, further four factors should be measured in detail by a known method. become.

When diluting and measuring blood, plasma, or the like, if the lot of the diluting reagent changes, the correlation between the coagulation time and the amount of coagulation factor may not match.
Usually, it is necessary to remake a calibration curve for determining the above correlation for each lot of the diluting reagent. Further, according to the method of the present invention, if it is from undiluted to about 1000-fold diluted,
It can be easily measured, and the effectiveness of the method of the present invention becomes more remarkable as the dilution ratio increases.

[0012]

According to the method of the present invention, the measurable upper and lower limit concentrations can be greatly expanded as compared with the conventional method, and the measurement in the low concentration range, which was conventionally impossible to measure, can be performed. Since it will be possible, the range of clinical use such as measurement with a quantitative sample will be expanded, and improvement of medical technology can be expected.
In addition, since the present invention has almost no room for intervention of human judgment as compared with the conventional method, rapid and accurate automatic determination of the coagulation factor becomes possible.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between heat generation sensor temperature and time during TT time.

FIG. 2 Plasma concentration (V corresponding to TT time and amount of coagulation factor)
Graph showing the correlation of NC)

FIG. 3 is a graph showing a correlation between APTT time and plasma concentration corresponding to the amount of coagulation factor.

FIG. 4 is a graph showing the relationship between heat generation sensor temperature and time during APTT time.

Claims (2)

[Claims] 1. A heat generating sensor, which has a heat generating effect and can measure its own temperature, is brought into thermal contact with blood, blood serum, blood plasma, or a diluted solution thereof, and heat is generated from the addition of a coagulant to the coagulation. A method for quantifying a blood coagulation factor, which comprises measuring a coagulation time from a temperature change of a sensor and quantifying the amount of the coagulation factor in the blood, serum or plasma from the correlation between the time and the amount of the coagulation factor. 2. The correlation between the coagulation time and the amount of coagulation factor is determined by changing the concentration of a dilute solution of blood, serum or plasma.
The coagulation time corresponding to the dilution series is measured using a heat generation sensor that has an exothermic action and can measure its own temperature.
The method for quantifying a blood coagulation factor according to claim 1, wherein a calibration curve showing a correlation between the coagulation time and the amount of the coagulation factor is prepared and the coagulation factor is quantified by the curve.