JPH043505B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to a blood coagulation measuring method and a blood coagulation measuring apparatus.

Structure of conventional examples and their problems Conventionally, bleeding time and whole blood coagulation time tests have been performed as tests for bleeding disorders, but recently, prothrombin time (PT) has been used as a test for the reaction process of blood coagulation. , partial thromboplastin time (PTT), activated partial thromboplastin time (APTT) and fibrinogen concentration (Fbg)
The measurement of the functions of extrinsic coagulation, intrinsic coagulation, and fibrin formation has become routine. Of these, fibrinogen quantification is the most complicated and the testing cost is high.

The methods for measuring each of the above-mentioned items are roughly as follows.

PT: Prewarm the calcium-enriched tissue thromboplastin reagent at 37°C, add plasma to it, and measure the time until it coagulates.

PTT (APTT): A mixture of partial thromboplastin reagent (activated partial thromboplastin reagent) and plasma is heated at 37°C for a certain period of time, and pre-warmed calcium chloride solution is added to solidify it. Measure the time it takes.

Fbg: Prepare fibrinogen standard solution (fibrinogen concentration known) diluted in several stages with Orenveronal buffer. Add thrombin reagent to each diluted fibrinogen standard solution and measure the time until coagulation. Plot the fibrinogen concentration and the obtained clotting time on a log-log graph and draw a calibration curve. Add a thrombin reagent to a 10-fold dilution of test plasma of unknown concentration, measure the clotting time in the same way, and derive the fibrinogen concentration from the standard curve.

As mentioned above, in the measurement of fibrinogen concentration (Fbg), operations such as plasma dilution and preparation of a calibration curve are complicated, and the measurement takes time. Furthermore, the reagents used are expensive, and the overall testing cost is quite high.

Blood coagulation tests are often performed when performing medical treatment where bleeding is expected, and testing as quickly as possible is necessary. The reality is that the demand for inspections has not been met.

Purpose of the Invention In view of the above circumstances, the first invention aims to provide a method for rapidly measuring blood coagulation, and the second invention aims to provide a method for rapidly measuring blood coagulation.
An object of the invention is to provide a device that can quickly and accurately measure blood coagulation.

Structure of the Invention The method for measuring blood coagulation of the first invention includes a process of mixing plasma and a coagulation reagent for measuring prothrombin time, partial thromboplastin time, or activated partial thromboplastin time, and irradiating the mixed liquid with light. capturing the scattered or transmitted light, converting it into an electrical signal, and recording changes in the level of the electrical signal in relation to the passage of time from the initial state at the time of mixing the plasma and coagulation reagent to the final stable state; Based on the recording result of the electrical signal, the prothrombin time or the time required for the level of the electrical signal to reach a predetermined percentage of the final stable state level from the time of mixing the plasma and coagulation reagent based on the initial state level is calculated. A process of determining a value corresponding to partial thromboplastin time or activated partial thromboplastin time, and determining the total amount of change in the level of the electrical signal from the initial state to the final stable state based on the recording result of the electrical signal, and determining the total change in the level of the electrical signal from the initial state to the final stable state. This process includes the step of determining a value corresponding to the fibrinogen concentration by multiplying the amount by a certain coefficient.

The reagent for measuring PT and Fbg in one operation is the above-mentioned PT measurement reagent, and the reagent for measuring PTT and Fbg in one operation is the above-mentioned PTT.
It is a reagent for measurement, and the same applies to the case where APTT and Fbg are measured in one operation. Fbg
It has been confirmed through experiments that substantially the same value is obtained when measured together with PT, PTT, or APTT.

According to the method of the first invention, light and electrical signals are used to immediately detect PT or PTT based on a change in the electrical signal.
Alternatively, APTT and Fbg can be determined, and two types of measurements can be performed in one operation, making it possible to significantly reduce time compared to conventional methods.

Moreover, the blood coagulation measuring device of the second invention is
As shown in the figure, there is provided a cell a into which plasma and a coagulation reagent for measuring prothrobin time, partial thromboplastin time or activated partial thromboplastin time are injected, means b for stirring the liquid content of cell a, and a means for stirring the liquid contained in cell a; a light emitter c that irradiates light to a
, a photoelectric converter d that receives scattered light or transmitted light from the cell a and converts it into an electrical signal, an A/D converter e that converts the electrical signal into a digital signal, and a microcomputer MiC, etc. The devices used include means _f1 for storing the value of the digital signal from the A/D converter e in association with the passage of time from the initial state at the time of mixing the plasma and coagulation reagent to the final stable state; a means _f2 for determining whether the signal has stopped changing; and a means f2 for determining the stop of the change in the signal; means f ₃ for calculating as a value corresponding to time (PT) or partial thromboplastin time (PTT) or activated partial thromboplastin time (APTT) and the total change from the initial state value to the final steady state value of said digital signal; means f ₄ for calculating the value corresponding to the fibrinogen concentration (Fbg) by multiplying this total amount of change by a certain coefficient, and displaying the values obtained by each of the calculation means f ₃ and f ₄ . It is equipped with a means g to do this.

Description of Examples Constant temperature reaction cell a into which plasma and coagulation reagent are injected
is set in the thermostatic chamber 1 as shown in Fig. 2, but before it is set, the coagulation reagent is added by pressing the push button 3 of the pipette 2. Plasma and coagulation reagent are instantaneously mixed by stirring means b. The pipette 2 is provided with a switch 4 which is activated when a push button 3 is pressed. On the other hand, the stirring means b is composed of a rotating body 10 made of an elastic material such as rubber and a motor 11 having a shaft that fixes the rotary body eccentrically. Therefore, when the cell a is pressed against the rotating body 10 and the motor 11 is rotated at high speed, the lower part of the cell a rotates at high speed, and a stirring effect similar to that obtained by shaking can be obtained.

As the light emitting device c, for example, a light emitting diode, an incandescent lamp, a laser, etc. are used. As the photoelectric converter d, for example, a photodiode, a phototransistor, a CdS element, etc. are used. The photoelectric converter d is provided at a position where the transmitted light from the cell a does not enter, but only the scattered light from the cell a enters.
A microcomputer MiC connected after the interface 12 having the A/D converter e includes a controller 5, an arithmetic unit 6, a memory 7, and a clock generator i.
etc. A numeric display 8 and a printer 9 are used as the display means g. The microcomputer MiC sends the set temperature to the temperature control circuit 14 of the heater 13 of the thermostatic chamber 1. It also sends an on signal to the motor control circuit and an off signal after the timer time has elapsed.

We will explain the operation when measuring prothrombin time (PT) and fibrinogen concentration (Fbg).

First, a calcium-enriched tissue thromboplastin reagent is preheated at 37°C, plasma is added thereto, and the reagent is placed in a thermostatic reaction cell a. At this time, the controller 5 receives a start signal indicating the zero point.

Monochromatic light is input into the cell a from the light emitter c, that is, a light emitting diode (LED), and the scattered light is received by the photoelectric converter d, that is, the photodiode.
The intensity of the scattered light is converted into an electrical signal whose intensity corresponds to the degree of blood coagulation (suspension). This electric signal is an analog signal, and after being amplified by an amplifier h, it is converted into a digital signal by an A/D converter e, and is input to the microcomputer MiC.

The microcomputer MiC receives a start signal from the switch 4 of the pipette 2, starts a timer in the device, and at the same time sends an output signal to the motor 11 for a fixed short period of time to start the rotating body 10.
During this period, cell a is rotated at high speed.
0, the plasma in cell a and the coagulation reagent are mixed instantly.

The controller 5 includes a clock generator i (clock generator) which is started by the start signal.
In cooperation with the memory device 7 and the arithmetic unit 6, the printing device 9 is driven to perform plot printing according to the output of the photoelectric converter d from the time when the plasma and the coagulation reagent are mixed. An example of the printed plot is shown in FIG.

The output does not change at a certain level until the reaction progresses and fibrin is formed at point A. Thereafter, the scattered light suddenly increases, passes through point B, and reaches point C, where the increase stops. This change is actually quite drastic.

Almost mid-height (if point A is 0% and point C is 100%)
40% point B), that is, a predetermined percentage of the final stable state value (40% in this example) based on the initial state value of the digital signal from the time when the digital signal input starts as described above. The time required to reach this point is the prothrombin time (PT).

Calibrate the voltage output difference H between points A and C, that is, the total amount of change from the initial state value of the digital signal to the final stable state value, by multiplying it by a certain coefficient k. The fibrinogen concentration (Fbg) is displayed on the numerical display 8 together with the aforementioned prothrombin time (PT) T. In addition,
The actual value of the voltage output difference H between points A and C above is:
Differences in numerical composition (what kind of light emitter, photoelectric converter, and amplifier are used)
It varies depending on the unit, and it also depends on how the unit is taken, so it is not uniquely determined. Therefore, the above-mentioned constant coefficient k is not uniquely determined either.
For this reason, after configuring the measuring device, the voltage output difference H is measured by measuring sample plasma having a known fibrinogen concentration, and the coefficient k is calculated based on this.

FIG. 4 shows a functional flowchart for the microcomputer MiC mentioned above. FIG. 5 shows a flowchart of the execution program. This will be explained below.

Initialize in step (1), wait for input of a start signal from switch 4 of pipette 2 in step (2), and start the clock in step (3) if there is an input. Next, input the sample clock in step (4). In step (5), add 1 to the clock count. In step (6), data V() is read from the A/D converter e. V() is the input voltage value in clock counting. In step (7), data V() and clock count are transferred to memory. In step (8), a drive signal is sent to the display means g.

In step (9), the deviation of the input voltage value V()
-V(-1)=ΔV is calculated, and in step (10) it is determined whether the deviation ΔV is 0 or not. In other words, it is determined whether or not point C in FIG. 3A has been reached.
If NO in this judgment, the process returns to step (4) and data is read, displayed, and stored again. YES
If so, move on to step (11) and set V() to the specified value.
A discrimination is made as to whether V is greater than or equal to ₀ , that is, whether it is before point A or has reached point C in FIG. 3A. If NO, return to step (4); if YES, that is, if point C has been reached, proceed to step (12).

In step (12), the deviation H between the final value V(N) and the initial value V(1) is calculated, and in step (13) the deviation H is calibrated. In other words, Hs is obtained by multiplying by a constant coefficient K. This value Hs is the fibrinogen concentration (Fbg). In step (14), the fibrinogen concentration Hs is output to the numerical display 8 for display. Next, the time t _n corresponding to 40% of the deviation H
To find , first, in step (15), calculate V
(1)+(40/100)×H=Vm, then in step (16), calculate the two values V closest to Vm that sandwich Vm.
Read (K)·V(K+1) from the memory table. In step (17), calculate the proportional distribution of Δ in Figure 3B by {Vm-V(K)}/{V(K+1)-V(K)}=Δ
In step (18), the prothrombin time T is determined by the calculation (K+Δ)×to=T. Here to is the clock period. In step (19), the prothrombin time T is output to the numerical display 8 and displayed. Then, print the data obtained in the last step (20).

In addition, prothrombin time (PT) is 40% of H.
The time taken is based on experience.
However, in Figure 3A, the rise from point A to point C is actually quite steep, and as a practical matter,
There is very little error between 10% and 90% of the time. That is, as a specific numerical value of the predetermined percentage in the claims, any value in the range of 10% to 90% can be selected.

Also, partial thromboplastin time (PTT)
and activated partial thromboplastin time (APTT)
It has been experimentally confirmed that the fibrinogen concentration (Fbg) value Hs (=k·H) in the measurement of prothrombin time (PT) is almost the same as the value in the measurement of the prothrombin time (PT). The rise from point A to point C was similarly steep.

Figure 6 shows an example of the correlation between fibrinogen concentration and prothrombin time (PT) measurement using the conventional method and the voltage deviation H shown in Figure 3A. An example of correlation with voltage deviation H in APTT) measurement is shown. From other follow-up tests, it was confirmed that the H value has sufficient validity as a fibrinogen concentration value. The formula for the regression line in Figure 6 is y=0.23・X+18.95, the correlation coefficient is r=0.855, and the formula for the regression line in Figure 7 is y=0.19・X+6.88, and the correlation coefficient is r= It is 0.901.

In addition, as shown by the imaginary line in FIG. 2, the photoelectric converter d may receive transmitted light from the cell a. In this case, the graph corresponding to FIG. 3 will be a graph in which the signal continues at a constant high level, then falls sharply, and then reaches a constant low level.

Effects of the Invention According to the method for measuring plasma coagulation of the first invention, prothrombin time (PT), partial thromboplastin time (PTT), or activated partial thromboplastin time can be detected immediately from a change in the electrical signal using light and electrical signals. (APTT) and fibrinogen concentration (Fbg) can be measured, but reduction has the advantage that two types of measurements can be quickly performed with just one operation. Further, according to the blood coagulation measuring device of the second invention, there is an effect that the measurement value can be determined extremely accurately using the measurement method according to the first invention.

[Brief explanation of drawings]

FIG. 1 is a block diagram showing the configuration of the second invention;
Figure 2 is a configuration diagram of the embodiment, Figure 3A is a time/output correlation graph, Figure 3B is an enlarged view of the vicinity of point B in Figure 3A, Figure 4 is a functional flowchart, and Figure 5. is a flowchart of the execution program, FIG. 6 is a correlation graph between H and fibrinogen concentration when measuring prothrombin time, and FIG. 7 is a correlation graph between H and fibrinogen concentration when measuring activated partial thromboplastin time. a... Cell, b... Stirring means, c... Light emitter,
d...Photoelectric converter, e...A/D converter, f ₁ ...
Storage means, f ₂ ... Signal change stop judgment means, f ₃ , f ₄
...Arithmetic means, g...Display means.

Claims (1)

[Claims] 1. A process of mixing plasma with a coagulation reagent for measuring prothrombin time, partial thromboplastin time, or activated partial thromboplastin time, and irradiating the mixture with light to capture scattered or transmitted light. a process of converting this into an electrical signal and recording changes in the level of this electrical signal in relation to the passage of time from the initial state when plasma and coagulation reagent are mixed until reaching a final stable state; and the recording result of the electrical signal. Based on the prothrombin time, partial thromboplastin time, or activation portion, the time required for the level of the electrical signal to reach a predetermined percentage of the final stable state level based on the initial state level from the time of mixing the plasma and coagulation reagent. Based on the process of obtaining a value corresponding to the thromboplastin time and the recording result of the electrical signal, the total amount of change in the level of the electrical signal from the initial state to the final stable state is determined, and this total amount of change is multiplied by a certain coefficient. A method for measuring blood coagulation, comprising the step of determining a value corresponding to a fibrinogen concentration. 2. A cell into which plasma and a coagulation reagent for measuring prothrombin time, partial thromboplastin time, or activated partial thromboplastin time are injected, a means for stirring the solution in this cell, and a light emitter for irradiating the cell with light; a photoelectric converter that receives scattered light or transmitted light from the cell and converts it into an electrical signal; an A/D converter that converts the electrical signal into a digital signal; and a digital signal from the A/D converter. means for storing the value in association with the passage of time from the initial state when plasma and coagulation reagent are mixed until reaching the final stable state; means for calculating the time required to reach a predetermined percentage of the value of the digital signal as a value corresponding to the prothrombin time, partial thromboplastin time, or activated partial thromboplastin time, and a final stable state value from the initial state value of the digital signal; means for calculating a value corresponding to the fibrinogen concentration by calculating the total amount of change up to and multiplying this total amount of change by a certain coefficient, and means for displaying the values obtained by each of the calculation means. Blood coagulation measuring device.