JPH0223825B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for optically measuring blood coagulation. Blood coagulation tests are extremely important tests for the treatment of patients with bleeding tendencies or for the follow-up management of patients undergoing anticoagulant therapy.
It is also an essential test prior to surgery.
One such coagulation test is the prothrombin time (hereinafter referred to as
Tests that measure the PT (abbreviated as PT) and activated partial thromboplastin time (hereinafter abbreviated as APTT) are well known, and are performed as comprehensive tests for the extrinsic and intrinsic coagulation mechanisms, respectively. . In addition, many other tests are used to diagnose and treat hemophilia and the like, including qualitative factor tests and quantitative factor tests that measure which coagulation factors are deficient and to what extent.

Each of these tests is performed using a dedicated reagent, but in each case, many types of clotting factors in the test sample (usually plasma from which blood cells have been separated are used) are detected by each reagent. The measurement is based on the phenomenon in which fibrinogen is activated by the action of fibrinogen and interacts with each other, eventually converting to insoluble fibrin, and the fluidity of blood is lost.

For example, in measuring PT, a reagent containing tissue thromboplastin as a main component is used, and the time from the time when the reagent is mixed with plasma, which is a test sample, until the test plasma begins to coagulate is measured. If the measurement time obtained for the test plasma is delayed compared to the similar measurement time for normal plasma (approximately 12 seconds, depending on the reagent and measurement technique used),
An abnormality in plasma factor, factor 1, factor 1, or factor 1 is suspected. Furthermore, by adding appropriate correction reagents and special operations and measuring the clotting time in the same way, it is possible to know which factor is deficient and to what extent. In both measurements, the principle is common that coagulation ends when fibrinogen in the test plasma is converted and precipitated into fibrin.

Furthermore, regarding APTT measurements and other coagulation tests, the phenomena used to determine coagulation are no different from PT, only the reagents used are different.

As mentioned above, the measurement of blood coagulation can be translated into the detection of fibrin formation and precipitation. Various specific methods have been proposed in the past, but they are extremely important as they greatly influence the reliability of coagulation tests.

The most basic method is to judge fibrin deposition with the naked eye and measure the coagulation time with a stopwatch, but this requires a high level of skill, and there are concerns about reliability due to individual differences among the measurers. Furthermore, methods of applying mechanical vibration and measuring electrical resistance have been proposed, but the former constantly applies unnatural stress while the coagulation reaction is progressing, so there are doubts about the accuracy of the measured values. In the latter case, there are problems with the influence of the probe inserted as a foreign object and contamination between samples. Further, all of the above-mentioned methods require a great deal of effort for continuous processing in response to the remarkable increase in the number of specimens in recent years, and are unsuitable for rapid processing of a large number of specimens.

Therefore, various mechanisms have been proposed today that utilize the phenomenon that light transmittance fluctuates during fibrin deposition and optically detect this phenomenon. These optical detection methods allow measurement in a natural state without contacting the test sample without affecting the coagulation mechanism, and are suitable for rapid processing of a large number of samples. The change is small, and therefore the amount of light transmitted by coagulation shows only a gentle fluctuation between before and after, which is insufficient in terms of coagulation detection sensitivity. In particular, when measuring blood with abnormalities in coagulation factors, the signal at the time of fibrin deposition is weaker than with a nozzle that does not use a coagulation reaction, making accurate determination of coagulation even more difficult. (processing, differential processing, combinations thereof, etc.) are not satisfactory. Furthermore, since transmitted light is used, it is completely impossible to measure highly turbid specimens that commonly occur depending on the pathological condition.

In view of the above-mentioned drawbacks, the present inventors conducted preliminary research and found that when detecting the scattered light generated when laser light is irradiated to fibrin that precipitates as the blood coagulation reaction progresses, it is difficult to detect the scattered light caused by fibrin precipitation. The inventors have discovered that there is an angle at which changes in intensity can be selectively detected, and have completed the present invention.

That is, the present invention provides a method for optically measuring blood coagulation, which is performed by irradiating fibrin precipitated in a sample liquid as a blood coagulation reaction progresses with a laser beam and detecting the scattered light generated thereby. This is an optical method for measuring blood coagulation, characterized in that it is carried out by detecting backscattered light among scattered light.

The present invention can be easily implemented, for example, as follows.

That is, for example, blood coagulation is measured by irradiating test blood or a mixture of plasma and reagent with a laser beam and measuring changes in the intensity of backscattered light to determine coagulation time and coagulation mode.

The present invention uses an optical detection method like the conventional method for measuring blood coagulation, but unlike the conventional method that uses transmitted light, it uses scattered light, and particularly backscattered light among the scattered light. is the scattering angle θ
= 30° to 60°, and provides a detection method that is more sensitive than conventional methods and is effective even for highly turbid samples. FIG. 1 is a record of changes in scattered light intensity A over time associated with solidification using the method of the present invention. In the figure, part a (scattered light intensity As) shows that the coagulation reaction is progressing due to mixing of the test sample and reagent, but no fibrin precipitation is observed yet, and part b shows that fibrin precipitation is active. Progressing state, part c (scattered light intensity
Af) is understood to be the state in which the conversion and precipitation of fibrinogen to fibrin has been completed, and it is empirically known that the time point Tc when transitioning from a to b is the start point of fibrin precipitation. That is, detection of coagulation corresponds to detection of part b, and the clearer the difference between state a and state c before and after coagulation, the more sensitive the measurement can be.

In measurements using transmitted light, a curve similar to that shown in Figure 1 (however, the direction of increase and decrease is opposite) is obtained for the amount of transmitted light before and after fibrin deposition, but as mentioned above, the difference in the signal after solidification is measured before and after solidification. is much smaller than in the case of backscattered light, and in plasma exhibiting coagulation abnormalities or highly turbid plasma, the transmitted light amount signal does not show any change even though coagulation is confirmed with the naked eye. The present invention is an optical method for measuring blood coagulation by measuring changes in the intensity of backscattered light, preferably at a scattering angle θ = 30° to 60°, and a method for measuring the amount of transmitted light. It is possible to obtain superior coagulation detection sensitivity compared to the conventional method.

FIG. 2 shows one embodiment of the arrangement of a laser light source, a test sample serving as a light scattering source, and a photodetector according to the present invention. A mixture 4 of a test sample and a reagent in the reaction cuvette 2 is irradiated with a constant amount of laser beam emitted from the laser light source 1, and the incident beam is scattered depending on the state of the mixture. A photodetector 3 that measures the intensity of this scattered light and converts it into an electrical signal is placed at a specific position relative to the reaction cube 2. The laser light source 1 is, for example, an oscillating light source.
A helium neon laser with a wavelength of 632.8 nm is preferred, the reaction cube 2 is a test tube cube with an internal diameter of 5 mm, and the photodetector 3 is a normal photoelectric conversion element. Assuming that the angle between the laser optical axis passing through the center of the reaction cube 2 and the photodetector 3 is θ, the intensity of the scattered light shows different dependence on the scattered light detection angle θ depending on the state of the light scattering source. FIG. 3 shows the intensity of scattered light from a mixture of sample plasma and reagent before coagulation recorded against θ using the apparatus of the above embodiment. Although this is a simplified illustration of several small peaks, it is characteristic that the intensity of scattered light increases in the direction where θ is large, that is, forward scattering. Intensity of scattered light before solidification
The scattered light intensity Af of As after solidification is such that the change due to fibrin precipitation (Af - As) is superimposed on As. On the other hand, as the coagulation detection sensitivity, that is, the ratio of the change in the intensity of scattered light before and after the coagulation reaction, the ratio of the change in the intensity of scattered light due to fibrin precipitation to the intensity of scattered light before coagulation R = (Af - As )/
FIG. 4 shows the relationship between R and θ for the above-mentioned embodiment, taking As. According to this, it can be seen that there is an optimal detection angle range in which the coagulation detection sensitivity is the highest. Differences in the mixture of the test sample and reagent, that is, the presence or absence of coagulation factor abnormalities and the level of turbidity, affect the scattering angle characteristics of the intensity of scattered light before coagulation shown in Figure 3; It does not have much influence on the optimum detection angle range, which is known from the scattering angle characteristics of detection sensitivity shown in . As an example, FIG. 6 shows the results of measuring the R value at a predetermined scattering angle using two types of hyperlipid plasma with high turbidity and plasma with normal turbidity as specimens. 6th
In the figure, -△- indicates plasma with normal turbidity;
-○- indicates the results when high-lipid plasma was used as the sample, and -●- indicates the results when high-lipid plasma was used as the sample. As is clear from this result, when high-lipid plasma with high turbidity is used as a sample, the R value may be 0 when θ is 90° or more (that is, when forward scattered light is measured). On the other hand, when measuring with backscattered light, it is found that the R value can be measured for any specimen, especially when θ is 30° to 60°. In other words, fibrin precipitated by blood coagulation is detected by backscattered light.
Preferably, by detecting at a scattering angle θ of 30° to 60°, the R value can be measured regardless of whether the sample has turbidity or not. Therefore, using a photodetector placed in this angular range,
Any sample for which a coagulation test is required can be measured with the best detection sensitivity, which is directly linked to the accuracy of the coagulation test. Prior to the disclosure of the technology of the present invention, there has been no mention of selectively detecting blood coagulation signals with the best sensitivity based on the study of the spatial distribution of the intensity of scattered light. In particular, the unique feature of the present invention is that it is possible to measure whole blood samples that do not separate blood cells, which could not be measured at all using conventional optical measurement methods, using similar backscattered light. This is what I did. This can only be achieved by using a laser light source as a light source and by knowing that only the detection angle with the highest sensitivity can be selected. Measurement using whole blood eliminates the enormous amount of labor involved in separating blood cells and the complexity of operations, allowing for rapid processing of a large number of samples, and also reducing the burden on the amount of blood sampled from the patient being tested. It's important.

FIG. 5 is a block diagram of an actual blood coagulation measuring device according to the present invention. The intensity signal of the scattered light converted into an electric signal by the photodetector is amplified by a signal amplifier 5 and input to an A/D converter 6. The computer 7 processes the intensity signal of the scattered light converted into a digital quantity by the A/D converter and determines the coagulation point. Conventionally, the determination of the freezing point has been performed using the fibrin deposition signal itself, its first derivative signal, second derivative signal, or a combination of these and other judgment criteria, and various programs have been created for each. Although it is well known that this is possible, it is not a limitation on the embodiments of the present invention. Furthermore, there is a display/print unit 8 for displaying data processed by a computer, a recorder 10 for continuously observing the coagulation process, and an automatic reagent dispensing mechanism/automatic sample preparation mechanism 9.Although not shown in the block diagram, The apparatus is configured with a reaction cuvette temperature control mechanism to maintain the same reaction conditions. As described above, it is clear that an automated and labor-saving blood coagulation measuring device can be easily realized by combining the present invention and existing techniques.

Although specific embodiments of the present invention have been described above, the present invention is not limited to these embodiments and can be applied to various specific embodiments within the scope of the present invention. be.

As described above, the present invention can measure only the change in the intensity of scattered light due to blood coagulation with the highest sensitivity, and thus provides a constant measurement method without affecting the coagulation mechanism, similar to conventional optical detection methods. However, it solves the drawbacks of low measurement sensitivity for test samples showing coagulation abnormalities and the difficulty of measuring high turbidity test samples, and it can be used for test samples that could not be measured conventionally. The automatic measuring device embodying the present invention can perform various coagulation tests quickly, accurately, and with high sensitivity.

[Brief explanation of drawings]

Fig. 1 is a time-scattered light intensity characteristic diagram of a mixed solution of a test sample and a reagent, Fig. 2 is a diagram of the principle of the photometry system according to the present invention, and Fig. 3 is a scattering light detection angle-scattered light intensity characteristic diagram before coagulation. , FIG. 4 is a scattering light detection angle-coagulation detection sensitivity characteristic diagram, and FIG. 5 is a block diagram of a blood coagulation measuring apparatus to which the present invention is applied. 1... Laser light source, 2... Reaction cube,
3...Photodetector, 6...A/D converter, 7
...computer. Figure 6 shows the results of measuring the R value at a predetermined scattering angle using two types of high-lipid plasma and plasma with normal turbidity as specimens, -△- indicates plasma with normal turbidity, - ○- indicates the results when high-lipid plasma was used as the sample, and -●- indicates the results when high-lipid plasma was used as the sample. Furthermore, R
The values are defined as follows. R=(Af-As)/As As; indicates the intensity of scattered light before the blood coagulation reaction of the specimen. Af: Indicates the intensity of scattered light after the blood coagulation reaction of the specimen.

Claims (1)

[Scope of Claims] 1. An optical method for measuring blood coagulation, which is carried out by irradiating fibrin precipitated in a test liquid as the blood coagulation reaction progresses with a laser beam and detecting the scattered light generated thereby, A method for optically measuring blood coagulation, characterized in that the method is carried out by detecting backscattered light among the scattered light. 2. The method for optically measuring blood coagulation according to claim 1, wherein the detection of backscattered light is performed at a scattering angle θ=30° to 60°. 3. The method for optically measuring blood coagulation according to claim 1 or 2, wherein the laser light source is a helium-neon laser light source with an oscillation wavelength of 632.8 nm.