JPH0311423B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a light scattering measuring device that can perform all measurements using blood coagulation reactions, antigen-antibody reactions, and immunoagglutination reactions.

[Background of the Invention] 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, and are also essential as tests prior to surgery. .

Such coagulation tests include prothrombin time (hereinafter abbreviated as PT) and activated partial thromboplastin time (hereinafter abbreviated as APTT).
There are well-known tests that measure the coagulation mechanism, and each is performed as a comprehensive test of the extrinsic and intrinsic coagulation mechanisms.

Furthermore, many types of measurement devices capable of measuring PT and APTT are now commercially available.

The blood coagulation reaction is considered to be a complex chain reaction involving coagulation factors, and if an abnormality is found in PT or APTT, it is necessary to perform a quantitative factor test to determine which factor is lacking to this extent. There is.

Conventionally, for factor quantitative testing, PT or APTT is measured using a correction reagent, qualitatively examining which factor is deficient, and then PT or APTT is measured using appropriate factor-deficient plasma. Without measuring the calibration relationship using the APTT test, it was not possible to determine which factors were deficient and to what extent, which required extremely complicated procedures and a great deal of effort. Furthermore, since this measurement method is indirect, the obtained results could not be said to be very accurate.

In recent years, blood coagulation tests are not comprehensive tests of coagulation reactions such as PT or APTT, but rather measurement of immune activity values using antigen-antibody reactions or biological activity values using enzyme reactions with synthetic substrates. According to
A method for directly quantifying each coagulation factor has been proposed.

According to this method, it is possible to perform simple, highly specific, and accurate measurements;
Since it is not possible to carry out measurements using this method using a measuring device for APTT, it is necessary to prepare a new expensive measuring device, which is extremely uneconomical.

In addition, on the contrary, the apparatus for performing measurements by this method has the disadvantage that it is impossible to perform so-called screening blood coagulation tests such as PT or APTT.

[Object of the Invention] The present invention was made in view of the above-mentioned circumstances, and provides a light scattering measuring device that can perform both blood coagulation tests such as PT or APTT and immunological measurements using antigen-antibody reactions. The purpose is to provide

[Structure of the Invention] The present invention provides a single laser light source and a system capable of independently measuring forward and backward scattered light generated as a result of the light irradiated from the light source entering a test liquid.
The above-mentioned photodetector, a means for differentially processing the electrical signal obtained from the photodetector, the maximum value of the signal obtained by the differential processing, and the time from the start of the reaction until the maximum value appears. This is an invention of a light scattering measuring device characterized by having a means for detecting.

That is, the present invention provides (1) installing one or more photodetectors capable of independently measuring forward and backward scattered light generated as a result of laser light incident on a subject, and (2) further installing the photodetector. By combining a means capable of differentially processing the electrical signal obtained from the device, a means for detecting the maximum value of the signal obtained by the differential processing, and the time from the start of the reaction until the maximum value appears, To provide a light scattering measurement device that can perform both blood coagulation tests such as PT or APTT and measurement of antigens and antibodies in a specimen using antigen-antibody reactions, which were impossible with conventional measurement devices. It is characterized by the fact that it makes it possible.

[Example] Preferred embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is an example of a schematic diagram of a light scattering measuring device according to the present invention, in which a laser light source 1, a reaction cuvette 2 for holding a test liquid 4, a photodetector 3 for measuring backscattered light, Photodetector 5, signal switch 6, signal preprocessor 7 for measuring forward scattered light
It is equipped with an A/D converter 8, a computer 9, a display/print unit 10 for displaying data processed by the computer, a reagent dispensing mechanism and an automatic sample preparation mechanism 11, a recorder 12 for continuously observing the reaction process, and the like.

For example, the laser light source used in the light scattering measuring device of the present invention has an oscillation wavelength of 700 to 800 nm.
Examples of the reaction cuvette include a test tube cuvette with an inner diameter of 5 mm, and examples of the photodetector include a common silicon photocell.

Next, an outline of the operation when performing a blood coagulation test or an antigen or antibody measurement using an antigen-antibody reaction using the measuring device of the present invention will be described below.

A laser light source 1 irradiates a reaction cuvette 2 holding a test liquid, which is a mixture of a sample and a measurement reagent, dispensed by a reagent dispensing mechanism and an automatic sample preparation mechanism 11. The intensity S of the resulting backscattered light or forward scattered light is detected by a photodetector 3.
or 5, and converts this into an electrical signal (hereinafter referred to as a light scattering intensity signal). Photodetector 3 or 5
Which of the light scattering intensity signals obtained by the above is used may be appropriately selected depending on the type of measurement, and which signal is to be sent to the signal preprocessor 7 is determined by the signal switch 6. Further, the light scattering intensity signal sent to the signal preprocessor 7 is electrically amplified and/or differentially processed. The data obtained as a result is input to the A/D converter 8 to be converted into a digital quantity, and this is input to the computer 9, which is subjected to appropriate arithmetic processing.
When performing a blood coagulation test, determine the point of coagulation;
In the case of measurement by antigen-antibody reaction, the concentration of antigen or antibody is calculated.

Note that the data processed by the computer 9 is displayed on the display/print section 10. The computer 9 also includes a reagent dispensing mechanism and an automatic sample preparation mechanism 1.
It goes without saying that the entire apparatus according to the present invention is controlled, including the control of 1, the reagent dispensing mechanism, and the automatic sample preparation mechanism 11, which recognizes the time when the sample and reagent are mixed as the reaction start time. stomach.

Further, the light scattering intensity signal obtained by either the photodetector 3 or 5 can also be output to the recorder 12 that continuously observes the reaction process. The data outputted to this recorder 12 is shown in FIGS. 2 and 3, for example.

Figure 2 shows the results of measuring the change over time in the light scattering intensity S due to the blood coagulation reaction when the angle θ between the laser optical axis passing through the center of the reaction cube 2 and the photodetector 3 is 50°. FIG. 3 shows the results of measuring changes over time in light scattering intensity S due to antigen-antibody reaction when the angle θ between the laser optical axis and the photodetector 5 is 155°. Incidentally, the angle meant by θ is illustrated in FIG.

In Figure 2, in part a, the coagulation reaction is progressing due to mixing of the sample and reagent, but no fibrin precipitation is observed yet, and in part b, fibrin precipitation is actively progressing. In this state, part c is fibrinogen (hereinafter abbreviated as FIB).
It is understood that this is the state in which the conversion and precipitation of 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. Therefore, it is possible to measure coagulation activity by determining Tc. Furthermore, blood clotting point
Detection of Tc can also be performed using other known methods that utilize scattered light.

Also, in Figure 3, the time T _A from the start of the reaction
Until then, the antigen-antibody reaction progresses due to the mixing of the sample and reagent, and an antigen-antibody complex is generated.After time T _A , the S value remains constant; in other words, the generation of the antigen-antibody complex is completed. It is well known that the situation is Therefore, measure this S value and use this value to calculate the S value immediately after mixing the sample and reagent.
By using the subtracted value (ΔS), it is possible to measure the concentration of the target antigen or antibody in the sample.

In addition, as a method of measuring the concentration of an antigen or antibody in a specimen using an antigen-antibody reaction, as shown in FIG. This may be performed using the change in (dS/dT) over time. More specifically, for example
The antigen or antibody concentration in the sample may be measured using the maximum value P of dS/dT.

Further, using the light scattering measuring device of the present invention, measurements using so-called agglutination reactions can be similarly performed. That is,
Latex particles immobilized with antigens or antibodies are reacted with the specimen, and the degree of aggregation is measured by backscattering intensity using a photodetector 3, and changes in light scattering intensity S as shown in Fig. 5 are measured. It is also possible to measure the target antigen or antibody contained in the sample by using the change in velocity (dS/dT) over time.

Examples of various measurements performed using the light scattering measuring device of the present invention are shown below.

[Measurement Example] Measurement Example 1 Measurement of PT (Sample) Citrated normal human plasma was appropriately diluted with physiological saline and used as a specimen.

(Measurement reagent) A thromboplastin reagent solution derived from rabbit brain was used.

(Operating Method) The following operations were performed using a light scattering measuring device having the configuration shown in FIG. In addition, the photodetector is θ=
A photodetector 3 fixed at a 50° position was used.

After placing 100μ of the sample, 100μ of the measurement reagent, and 100μ of the 0.02M calcium chloride aqueous solution in a test tube type reaction cuvette with an inner diameter of 5mm and mixing them well, use the above light scattering measuring device to measure the time at which the light scattering intensity S starts to increase. I asked for Tc.

(Results) The results obtained are shown in Figure 6. Furthermore, Figure 6 shows
The Tc value (seconds) obtained for each dilution rate of citrated normal human plasma on the horizontal axis is plotted along the vertical axis, and the points are connected.

As is clear from this result, it can be seen that a good calibration relationship can be obtained.

Measurement example 2 Measurement of blood coagulation factor (FIB) (sample) Standard plasma containing a specified concentration of FIB is dissolved in physiological saline.
The sample was diluted 61 times.

(Measurement reagent) A 12.5-fold dilution of rabbit-derived anti-FIB serum was used.

(Operating Method) The following operations were performed using a light scattering measuring device having the configuration shown in FIG. In addition, the photodetector is θ=
A photodetector 5 fixed at a position of 15° was used.

60 μ of the sample and 300 μ of the measurement reagent are placed in a test tube type reaction cuvette with an inner diameter of 5 mm, mixed well, and the light scattering intensity S1 immediately after mixing and the light scattering intensity S2 after being left at room temperature for 15 minutes are determined by the light scattering intensity described above. It was determined using a measuring device.

(Results) The results obtained are shown in FIG. Furthermore, Figure 7 shows
It is a graph that connects the points obtained by plotting the amount of change in light scattering intensity (S2-S1) obtained for each FIB concentration (mg/dl) on the horizontal axis along the vertical axis.

As is clear from this result, it can be seen that a good calibration relationship can be obtained.

Measurement example 3 Measurement of blood coagulation factor (FIB) (Part 2) (sample) The same one as in Measurement Example 2 was used.

(Measurement reagent) The same one as in Measurement Example 2 was used.

(Operating Method) The following operations were performed using a light scattering measuring device having the configuration shown in FIG. In addition, the photodetector is θ=
Measurements were made using the 50° position.

Place 60 μ of the sample and 300 μ of the measurement reagent in a test tube type reaction cuvette with an inner diameter of 5 mm, mix well, use the light scattering measurement device described above to determine the light scattering intensity S, and then differentiate this to obtain the dS/dT value. The maximum value P was determined.

(Results) The results obtained are shown in FIG. In addition, Figure 8 shows
P obtained for each FIB concentration (mg/dl) on the horizontal axis
It connects the points whose values are plotted along the vertical axis.

As is clear from this result, it can be seen that a good calibration relationship can be obtained.

[Effects of the Invention] As described above, the present invention allows two measurements that could not be performed with a single conventional device, namely, a measurement using a blood coagulation reaction and a measurement using an antigen-antibody reaction, to be performed with high precision. Moreover, this light scattering measuring device has the ability to be applied to measurements using immunoagglutination reactions, making it suitable for use in clinical laboratories. This invention greatly contributes to improving the efficiency of testing and rationalizing costs.

[Brief explanation of the drawing]

FIG. 1 shows a schematic diagram of a light scattering measuring device of the present invention. 1...Laser light source, 2...Reaction cuvette for holding test liquid 4, 3...Photodetector for measuring backscattered light, 4...Test liquid, 5...Forward scattering light Photodetector for measurement, 6...Signal switcher, 7...Signal preprocessor, 8...A/D converter, 9...Computer, 10...Display printing section for data processed by computer 9 , 11...
Reagent dispensing mechanism and automatic sample preparation mechanism, 12...
A recorder that continuously observes the reaction process. Figure 2 shows the change over time in the light scattering intensity S of the test liquid during the blood coagulation reaction, and the S value obtained for each time on the horizontal axis is plotted along the vertical axis. The plotted points are connected. Third
The figure shows the change over time in the light scattering intensity S of the test liquid during the antigen-antibody reaction, and the S value obtained for each time on the horizontal axis is plotted along the vertical axis. It connects the dots. Figure 4 shows
FIG. 2 is a principle diagram of the optical system of the light scattering measuring device of the present invention. Note that the numbers assigned to each part in the figure are the same as those in FIG. 1. Figure 5 shows the time course of the rate of change dS/dT of the light scattering intensity S of the test liquid in the antigen-antibody reaction, and the horizontal axis shows the dS/dT values obtained for each time. It connects the points plotted along the vertical axis. Figure 6 shows measurement example 1.
This is a calibration curve showing the relationship between the dilution rate of citrated normal human plasma and prothrombin time (Tc) obtained in . Figure 7 shows the fibrinogen concentration (mg/dl) obtained in Measurement Example 2;
This is a calibration curve showing the relationship with the amount of change in light scattering intensity (S2−S1). Note that S1 indicates the light scattering intensity immediately after mixing the specimen and the reagent, and S2 indicates the light scattering intensity after leaving it at room temperature for 15 minutes. Figure 8 is a calibration curve showing the relationship between the fibrinogen concentration (mg/dl) and the maximum value P of the dS/dT value obtained by differentiating the light scattering intensity S, obtained in Measurement Example 3. be.

Claims (1)

[Scope of Claims] 1. One laser light source; One or more photodetectors each capable of independently measuring forward and backward scattered light generated as a result of the light irradiated from the light source entering a test liquid; means for differentially processing the electrical signal obtained from the photodetector; means for detecting the maximum value of the signal obtained by the differential processing; and the time from the start of the reaction until the maximum value appears. A light scattering measuring device comprising: