JPS61215948A - Particle flocculation discriminating device - Google Patents

Abstract

PURPOSE:To make possible the quick and exact automatic discrimination of flocculation by disposing a TV camera and light sources on the same side of a microplate and taking in the optical state of the microplate by the TV camera. CONSTITUTION:An image taking-in device 1 is provided with a plate stage 4 which is formed of a light reflective material and on which the microplate is imposed in the lower part thereof. The stage 4 is so formed that the light transmitted through the microplate is reflected and can be made incident again on the microplate 3 if the microplate 3 to be imposed thereon is transparent. The solid-state element TV camera 6 and the two light sources 7 near the same are provided above the stage 4. The light sources 7 irradiate light to the microplate 3 and take the refracted light thereof as the information on the optical state into the TV camera 6. The inputted image signal is processed and the flocculation state is discriminated in an image processing unit 2.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an apparatus for determining particle aggregation in clinical tests, and more particularly to an apparatus for determining agglutination in microtiter methods.

(Prior Art) Currently, the microtiter method in immunoassay is widely used to detect the presence or absence of agglutination on a microplate and to measure trace amounts of immune components. The presence or absence of these aggregations is determined visually with the naked eye.In this visual judgment, the presence or absence of aggregation is determined as the area of the area below the brightness that constitutes the distribution of particles in the well, and standard aggregation A? Turn, standard non-cohesive A? Judgments are made by comprehensively combining judgments such as comparison with the turn and consideration of the relationship with the appearance of adjacent wells. Therefore,
Visual judgment requires a high degree of skill, and since it is an intuitive testing method, there are individual differences between judges, and there are also drawbacks such as a lack of reproducibility even among the same judge.

Automating this visual judgment using equipment is not a fool's errand that will lead to labor savings, but it will also give objectivity to the judgment results and can be expected to greatly improve measurement accuracy.

Although the particle agglutination reaction has many applicable test items, is easy to operate, has high detection sensitivity, and is suitable for processing large amounts of samples, the only drawback is that the final judgment cannot be automated. there were. Therefore, solving this drawback and developing a highly accurate automatic determination method for agglutination reactions is extremely important for clinical testing and will greatly contribute to the development of medical science.

(Problem to be solved by the invention) However, it would be difficult to implement the same judgment as the visual judgment based on a complex combination of factors, which is currently being done, because the equipment would be extremely complicated and economically expensive. It is of little use. Therefore, in automatic determination by equipment, the technical point is to limit the number of determination factors and how to match the results of visual determination. All of the automatic determination devices that have been attempted so far have either used a photometer to correlate the absorbance at the center of each well with aggregation, or have tried to correlate the light intensity ratio between the center of the well and the periphery of the well to determine aggregation. There have been attempts to make it compatible, but all of them have the ability to only serve as an auxiliary means to visual judgment, and none have the ability to replace visual judgment.

In addition, since conventional equipment measures wells one by one,
Measurements take time and require a plate moving device.

Further, conventional automatic determination devices utilize transmitted light, but this method can only achieve extremely insufficient measurements, as will be explained below.

That is, a sample for a particle aggregation reaction contains various proteins such as serum, and these components often precipitate separately from the particle agglutination reaction, making the entire solution cloudy.

As a result of careful observation of numerous tests and boars, the present inventors have found that this phenomenon is caused by extremely complex combinations of sample serum and reagent solutions, especially those that have been mixed for a long time, For the first time, we discovered that this phenomenon occurs so frequently that it cannot be ignored in foods with high salt concentrations.

However, in the conventional apparatus, no measures were taken to prevent this clouding, and only insufficient measurements were possible. In other words, when measuring transmitted light, the amount of light is significantly reduced due to diffuse reflection from precipitates, so the amount of transmitted light does not correspond to the particle aggregation reaction, and automatic aggregation determination equipment that converts particle concentration into brightness cannot accurately determine aggregation. could not.

Furthermore, in conventional devices, aggregation was determined by measuring transmitted light, so the transmitted light was absorbed by the particles only once when passing through the well from the bottom to the top, resulting in poor contrast. It was unclear.

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems and provide a particle aggregation determination device that is simpler than conventional devices and has higher accuracy than visual determination.

(Means for Solving the Problems) In order to achieve the above object, the present invention disposes a TV camera and a light source on the same side of a microplate, and captures the optical state of the microplate using the TV camera. This makes it possible to quickly and accurately automatically determine aggregation.

That is, it has a plate holder on which a microplate is placed, a TV camera and a light source arranged above the plate holder, and the plate holder allows the microplate to be placed to receive incident light from the light source. It is characterized by comprising an image capturing device configured to freely reflect the transmitted light when the transmitted light is transmitted below the plate, and an image processing device that processes the image signal input from the TV camera and determines the state of aggregation. Particle aggregation determination device.

The plate holder is used to place the microplate at a predetermined location, and when the microplate to be placed is transparent, it is designed to reflect the transmitted light of the microplate and re-enter the microplate. be done.

This is accomplished by forming the plate holder from a light-reflecting material or by providing a reflective member on the surface of the plate holder. Further, it is preferable that the plate stand is provided as close as possible to the bottom surface of the well of the microplate, and most preferably, it is in close contact with the bottom surface of the well. This prevents a decrease in the proportion of light that is complicatedly refracted by the microplate and is reflected by the plate stand and reaches the TV camera.

When the microplate is not transparent and is made to reflect light, for example, when the microplate itself is made of a light-reflecting opaque material, but when the transparent plate is coated with a light-reflecting opaque material, the plate holder will be able to reflect light. There is no need for it to be made reflective.

A TV camera is provided above this plate holder. This TV camera is for capturing the optical state of each well, and needs to be positioned as upwardly as possible to capture the optical state of all wells at once. A solid-state camera is preferable for this TV camera from the viewpoint of accuracy and stability of operation. TV camera lenses use lenses with little geometric distortion, and can also be equipped with filters.The color of this filter should be complementary to the color of the particles, so that the agglomerated images can be This is preferable because it can be efficiently distinguished from the other.

Further, a light source is also provided above the plate holder. This light source is used to irradiate light onto the microplate and capture the reflected light into a TV camera as information on the optical state. The light source is selected and used as appropriate, but a fluorescent light with a high color temperature is preferable because it can measure a variety of colors of particles, and a tranno-nutar inverter is used as a power source because it has less flicker and has good image reproducibility. It is preferred to use a high frequency fluorescent light with a

A milky white reflector may be provided around the plate holder. The purpose of this reflective plate is to prevent the center from becoming relatively bright and the periphery to become dark in a transparent microplate, and to make the brightness within each well substantially uniform.

An image processing device is connected to the TV camera.

This image processing device is a device for processing image signals input from a TV camera and determining the state of aggregation. This image processing device includes a video input section that receives the output from the TV camera, a part of the digital image memo IJ that records the data of each pixel, and a video output section that sends the contents to a computer as necessary. A microcomputer for calculating and processing the output, a monitor television for video input, and a printer for recording the calculation results are provided.

(Function) In the particle aggregation determination device of the present invention, a light source irradiates each well of a microplate with light of uniform intensity, and this irradiated light enters the diluent in the well. With this incident light, even if the diluted solution has cloudiness, the range of particle concentration that can be determined remains the same as the range of particle concentration when no cloudiness occurs.

On the other hand, the incident light passes through the diluted solution while being absorbed by the particles, is reflected by the bottom of the well or the reflecting plate, and passes through the diluted solution while being absorbed by the particles again. Therefore, the contrast of light passing through each part is emphasized depending on the density of the particles. The light emitted from each well is incident on a TV camera, which captures the optical state of all wells. This captured optical state is sent as an image signal to an image processing device, and this image processing device performs predetermined processing to determine aggregation.

(Example) Hereinafter, an example of the particle aggregation determination device of the present invention will be described based on FIGS. 1 and 2.

FIG. 1 is a diagram schematically showing a particle aggregation determination device, and FIG. 2 is an enlarged sectional view of the plate mounting portion of the same.
, - In FIG. 1, 1 is an image capturing device, and 2 is an image processing device. The image capture device 1 is provided with a plate mount 4 made of a light-reflecting material on which a microplate 3 is placed at the bottom thereof, and around the plate mount 4 there is a cross-sectional curve that widens upward. A reflecting plate 5 having a shape is provided. Further, a solid-state TV camera 6 is provided above the plate holder 4, and the TV camera 6 is equipped with a 16m5 TV lens as standard, and is further provided with a filter. Two light sources 7.7 are provided near the TV camera 6. The light sources 7, 7 are high-frequency fluorescent lights with a high color temperature. Further, a diffuser 8 made of a milky-white translucent plastic plate is provided between the light sources 7, 7 and the plate holder 4 in order to make the light from the light sources 7, 7 uniform.

The image processing device 2 includes a video input section 9 that receives the output of the TV camera 6, a digital image memory section 10 that records data of each pixel, and a video output section 11 that sends the contents to a computer as necessary. , and a microcomputer 12 for calculating and processing the output, and is further provided with a monitor television 13 and a printer 14.

Next, a method for automatically determining aggregation using the above particle aggregation determination device will be described.

(1) The microplate 3 for determining aggregation is installed on the microplate holder 4.

(2) Select the illumination 7 and TV left camera filters, adjust the grain, and capture the image.

(3) The captured image can be processed as it is, or it can be recorded on a 70-tup disk and loaded into the computer 12 as necessary for image processing and calculation processing.

Various types of image processing are possible, and one example will be described below.

■ Manually input signals from TV cameras into image memory.

■ Convert luminance 0 in the image to luminance 1...Convert the luminance gradation of the image data on memory. Luminance 0 is black and F is white.

(2) Superimpose mask images in which only the part inside the well of the microplate has a brightness of F and the other part has a brightness of O, and calculate pixel by pixel between the two images to synthesize a new image. This is because the inner part of the image captured by the TV left camera that contains necessary data is removed, leaving only the data where the agglomeration pattern exists.

■ Convert luminance 0 in the image to F. This leaves only the image of the well.

■ Convert luminance of 8 or higher in the image to F. (The brightness value is selected depending on the color tone, density, etc. of the particles to be measured.) (1) Convert brightness 1 to 8 in the image to brightness 0.

■ Overlapping screens to specify the order of image calculations Computer image calculations are performed in the order from top to bottom and left to right of the screen. That is, the calculations are performed in descending order of the y values, or if the y values are equal, the calculations are performed in the order of the smaller X values. That is, in screen A shown in Fig. 3, 2.4 from the left
, 1.3. Figure 4 shows screen B for specifying the order of image calculation, which is called screen A.
When superimposed, screen C shown in FIG. 5 is synthesized. The order of this calculation is 1.2, 3.4 from the left. This method is adopted in this device to match the order of wells and the order of calculation. Note that the area added by screen B is sufficiently small compared to the screen, and does not affect the determination of positive or negative.

■ Calculate the area of 96 patterns on the screen.

■ Compare with the set standard value and judge whether the agglutination reaction is positive or negative.

[F] Record the results.

Next, a description will be given of measurement results comparing the influence of cloudiness when measuring transmitted light and when measuring reflected light.

FIG. 6 shows the measurement results of the relationship between particle concentration and brightness when transmitted light was measured. In this figure, the relationship between the brightness of the well and the particle concentration in a sample that does not produce cloudiness is represented by ABC. AB is a region where particle concentration and brightness correspond, and BC is a region where particle concentration is high and light does not pass through, and where particle concentration and brightness do not correspond. K represents the amount of transmission reduced by diffused reflection due to turbidity generated within the well. Therefore, the relationship between brightness and particle concentration in a turbid well is expressed as DEC. As a result, the brightness in the well changes only in the OE region as the particle concentration changes, and the brightness does not change during 9 EB even if the particle concentration changes. In this way, when transmitted light is used, in a sample with precipitates, the area where changes in particle concentration cannot be measured expands, leading to errors in determining aggregation.

On the other hand, FIG. 7 shows the measurement results of the relationship between particle concentration and brightness when reflected light was measured. In the measurement of this reflected light, samples without white turbidity are expressed by ABC as in Figure 6, but samples with white turbidity are expressed as DE.
It becomes C'. As can be seen from this result, in the measurement of reflected light, the area where the brightness does not change does not increase with respect to the change in particle concentration due to clouding.

In the measurement using the apparatus shown in FIG. 1, the light passing through the well is reflected by the plate holder 4, and that light is measured as transmitted light. Therefore, the addition of figures 6 and 7 is $
When measuring with the apparatus shown in Figure 1, the influence of cloudiness is largely offset and virtually eliminated. For this reason, unlike the conventional technology that uses only transmission, the present invention is a measurement method that involves both reflected light and transmitted light, and this is a factor that allows stable judgments to be made even in turbid samples.

Next, the results of comparing the contrast between the case of measuring only transmitted light and the case of measuring both transmitted light and reflected light of the apparatus of the present invention will be explained. Figure i@8 shows the results of this measurement.

From this result, the contrast when only transmitted light is measured is part A, and the contrast when both transmitted light and reflected light is measured is part B. Therefore, when measuring both transmitted light and reflected light, In this case, the contrast is emphasized only in the C portion.

(Effects of the Invention) The present invention is configured as described above, and the optical state of the wells is captured using a TV camera, so that data of all wells in one microplate can be processed extremely quickly. Still, since the optical state of all wells is captured at once, f
No V-t moving device is required, and the operation can be simplified. Furthermore, it is possible to obtain not only information about the particle concentration in the well as a photometer, but also information about the two-dimensional spread9 of particle aggregation, which allows for a greater variety of types of information than when using a conventional photometer. Data can be obtained at high speed. Further, in the present invention, the light source and the TV camera are on the same side with respect to the microplate, and the reflected light and the light that enters the diluent in the well, is reflected by a plate stand, etc., and passes through the diluent again, and the light is collected and used for aggregation. Since the determination is made, the light is not affected by cloudiness and the contrast is enhanced, so that particle aggregation can always be accurately determined.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram of an embodiment of the particle aggregation determination device of the present invention;
Figure 2 is an enlarged cross-sectional view of the same plate placement part, Figures 3 to 5 are screens showing the image calculations, and Figure 6 is the relationship between particle concentration and brightness when measuring transmitted light. A diagram showing
FIG. 7 is a diagram showing the relationship between particle concentration and brightness when reflected light is measured, and FIG. 8 is a diagram showing the measurement results of contrast. l...image capture device, 2...image processing device, 3...
・Microplate, 4...Plate stand, 5...
Reflector, 6...TV camera, 7...-light source, 9...
Video input section, 10...Digital image memory part, 1
1.--Video output unit, 12.--Computer. Patent Applicant Fujirebio Co., Ltd. Agent Patent Attorney 1) Masahiro Naka 1st cause 3rd cause 4th cause A Crystal plane B 5th cause 6th figure 7th figure 7tJltf-L! Rinse 1 ki Figure 8

Claims (1)

[Claims] The plate mount includes a plate holder on which a microplate is placed, and a TV camera and a light source arranged above the plate holder. The invention is characterized by comprising an image capturing device that is configured to freely reflect the transmitted light when the transmitted light is transmitted, and an image processing device that processes the image signal input from the TV camera and determines the state of aggregation. Particle aggregation determination device