JPS62105031A - Decision device for particle flocculation - Google Patents

Abstract

PURPOSE:To decide the flocculation of particles accurately by providing a light source above and below a plate mount table and guiding both reflected light from the upper light source and transmitted light from the lower light source to a TV camera. CONSTITUTION:A particle flocculation decision device has the main light source 8 provided above the plate mount table 5 and the auxiliary light source 9 below it. The main light source 8 irrdiates each well of a microplate 6 with light having uniform intensity and the irradiated light is made incident on the dilution liquid in the well. The auxiliary light source 9, on the other hand, irradiates the reverse surface of the plate 6 with light having uniform intensity and the inside of the well is lighted brightly with the light from below. Then, light emitted from each well is made incident on the TV camera 13 through a mirror 14, a filter 15, and a mirror 14 and optical states of all wells are inputted to the TV camera 13 and those input optical states are sent as an image signal to an image processor 2, which performs specific processing to decide the flocculation. Thus, the flocculation is decided speedily and accurately.

Description

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

[Conventional technology]

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 by visual judgment using the naked eye, and in this visual judgment, the presence or absence of aggregation is determined as the area of the portion below the brightness that constitutes the distribution of particles within the well. In addition, judgments are made by comprehensively combining judgments such as comparison with standard agglutination t4 turn and standard non-aggregation t4 turn, and also considering the relationship with the state of adjacent wells. Therefore, visual judgment requires a high level of skill, and since it is an intuitive testing method, there are individual differences between judges, and there are also disadvantages 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.

Furthermore, automatically printing test results can completely eliminate errors in transcription of measurement results. 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 shortcoming and developing a highly accurate automatic determination method for agglutination reactions is extremely important for clinical testing and will contribute to the development of medicine.

[Problem that the invention seeks to solve]

However, it would be impractical to implement the same judgment as the current visual judgment based on a complex combination of factors because the equipment would be extremely complicated and economically expensive. Therefore, in automatic determination by equipment, the technical challenge is to limit the number of determination factors and how to match the result 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.

The inventors of the present invention have carefully observed a large number of test results, and have found that this phenomenon is caused by extremely complex combinations of sample serum and reagent solutions. For the first time, we discovered that this phenomenon occurs so frequently that it cannot be ignored when salt concentrations are high.

However, in the conventional apparatus, no measures were taken to prevent this clouding, and only insufficient measurements were taken. In other words, in the measurement of 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 fluid concentration into brightness cannot accurately determine aggregation. could not.

In addition, in order to solve the above-mentioned problems, the present inventors have proposed an apparatus that irradiates the upper part of a microplate with light and uses the reflected light and transmitted light to determine particle aggregation (
(Japanese Patent Application No. 60-57880).

However, although this device eliminated the effects of clouding, it was not possible to increase the amount of light in the wells of the microgrates enough to provide sufficient contrast using only illumination from above. In other words, in order to increase the amount of light in the well to a level that provides sufficient contrast, the amount of light from the light source must be extremely large, and a cooling device such as a cooling device that generates a lot of heat must be installed, making the device complex. And it was expensive. Moreover, the power consumption was also increased. Furthermore, since the dynamic range of the TV left camera is limited, the amount of light can only be increased until the upper surface of the microplate reaches a predetermined brightness, making it impossible to provide sufficient brightness within the well. Furthermore, an image of the light source appears on the liquid surface in the well, and this may overlap with the aggregation image, making measurement difficult.

It is an object of the present invention to solve the above problems and provide a particle aggregation determination device that is simpler than conventional devices and has higher accuracy than visual determination.

[Means for solving problems]

In order to achieve the above-mentioned object, the present invention arranges light sources on both the upper and lower sides of a microplate, and captures reflected light from the upper light source and transmitted light from the lower light source into the TV left camera. This improves both the effect of clouding and contrast, which could not be achieved with a light source, and enables quick and accurate automatic determination of aggregation.

That is, the particle aggregation determination device of the present invention includes a light-transmissive grating table on which a microplate is placed, a light source arranged above and below the plate table, and an upper side of the microplate placed on the plate table. an image capturing device having a TV left camera installed so that light emitted from the TV enters; and an image processing device that processes image signals input from the TV left camera to determine the state of aggregation. It is structured with the following characteristics.

The plate stand of the particle aggregation determination device of the present invention is for placing a microplate at a predetermined position. This grating stand is formed to be light-transmissive so that light from a light source provided below can enter the microplate. Therefore, at least the area on which the microplate is placed may be made of a transparent or semi-transparent body, and the area corresponding to the well may be cut out to provide through holes. It is preferable to use a translucent material such as a milky white plate so as to adjust the amount of incident light from the light source and to make the incident light uniform. Further, the plate holder may be provided so as to be movable inside and outside the image capture device. By providing a movable plate holder, microplates can be automatically and efficiently fed and discharged.

The microplates placed on this plate holder are of any type, size, number of wells, etc., but are formed to be light transmissive, that is, transparent or translucent. It requires that.

A main light source is provided above the plate holder.

This main light source is for irradiating light onto the micro grate and allowing the TV camera to capture the reflected light as information on the optical state. The main 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.Also, a transistor inverter is used as the power source because it has less flicker and has good image reproducibility. Preferably, a high frequency fluorescent light is used.

On the other hand, an auxiliary light source is provided below the grate stand. This auxiliary light source irradiates light from the back side of the microplate, and the transmitted light is captured by a TV camera as information on the optical state, and is used to correct the brightness within the well. The light quality of the auxiliary light source may be the same as the main light source, but
The amount of light is preferably about 1,000,000,000 yen, and the most preferable is about 1 of that of the main light source. Therefore, it is preferable that the auxiliary light source be able to adjust the amount of light as desired, and most preferably one that can adjust the amount of light without changing the color temperature of the light and without flickering.

Furthermore, a TV camera is provided so that the light emitted from the upper side of the plate stand, that is, the upper side of the micro-grate, is incident thereon. This TV camera is for capturing the optical state of each well and outputting an image signal to an image processing device. As long as this TV camera is far enough from the microplate to capture the optical state of all wells at once, the light may be bent through a mirror or the like and then incident on the TV camera. By providing the mirror, the TV camera can be placed at any position, and the device can be made more compact.

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.

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 to determine 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 memory that records the data of each pixel, a video output section that sends the contents to the computer as necessary, and this output. It consists of a micrograter that processes the calculations, a monitor television with video input, and a printer that records the calculation results.

A light amount adjustment plate may be provided between the main light source, the auxiliary light source, and the plate arrangement. This light amount adjustment plate is used to adjust the amount of light irradiated from the light source to the microplate and to irradiate the entire microplate with a more uniform amount of light, and is formed of a translucent material such as an opalescent plate. The light i' adjusting plate for the auxiliary light source can achieve the same effect by forming the grate holder with a semi-transparent material as described above.

Preferably, the TV camera is configured to capture only the light emitted from the microplate. That is,
A hood may be provided in the path of the light emitted from the microplate, or the entire device may be placed inside a box.These methods prevent unnecessary light from entering the TV camera, making measurements more accurate. Moreover, the contrast can be improved.

[For production]

In the particle aggregation determination device of the present invention, the main light source irradiates each well of a micrograte 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 auxiliary light source irradiates the bottom surface of the microplate with light of uniform intensity, and this light from below brightens the inside of the well, making it possible to brighten the inside of the well with a smaller amount of light than when irradiating light from above. In addition, when light is irradiated from above, the top surface of the microplate, which is flat and close to the light source, becomes extremely bright compared to the inside of the well, but when light is irradiated from below, the inside of the well becomes brighter than the top surface of the microplate. .

Therefore, the light from below the auxiliary light source improves the contrast compared to the light from above.

Then, the light emitted from each well enters the TV camera, and the TV
A camera captures the optical status 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〕

An embodiment of the particle aggregation determination device of the present invention will be described below with reference to FIGS. 1 and 2.

Figure 1 is a diagram showing the outline of the particle aggregation determination device;
The figure is an enlarged sectional view of the plate holder portion same as above. 1st
In the figure, reference numeral 1 is an image capturing device, and reference numeral 2 is an image processing device.

The image capture device 1 is formed of a sealed box 3 in a light-shielding manner, and inside thereof a movable plate holder 5 is provided on a non-reflective plate 4 made of a black plate. As shown in FIG. 2, this plate holder 5 has a light-transmissive portion 7 made of a milky white plate formed at the portion on which the micro-grate 6 is placed. A rack (not shown) is formed at one end of the grate holder 5, and is adapted to move forward and backward in mesh with a pinion (not shown) provided on the non-reflection plate 4.

A main light source 8 is provided above the grate holder 6, and an auxiliary light source 9 smaller than the main light source 8 is provided below. A light amount adjusting plate 10 made of a milky white plate is provided between the main light source 8 and the plate holder 51.
Furthermore, this light amount adjustment plate 10 and the non-reflection plate 4 are connected.
Seven boxes. □, □ 1 □□ □ 1 installed: Yes.

2. The corresponding part is paired with the microplate 6 of the light amount adjustment plate 1O, which has a substantially rectangular cylindrical shape and whose inner surface is painted black: 7
- A door □2 is provided. child. 7-do□2 is'
Wlrr: IJ□yo, wa, ya. □Yo, 7ka.

□: 13 are connected. Mirrors 14 and 14 are provided at the bends of the hood 12, and buttons are provided to allow the light from the microbe 6 to enter the TV left camera 3. Furthermore, the hood 12 is provided with an optical filter 15. The TV camera 13 is a solid-state TV camera equipped with a 24vrm camera lens.

The image processing device 2 includes a video input section 17 that receives the output of the TV left camera 3, a digital image memory section 18 that records data of each pixel, and a video output section that sends the contents to a computer as necessary. 19, a microcomputer 20 for calculating and processing the output, and a monitor television and a printer 22.

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

(1) Install a micrograte for determining aggregation on the microplate stand.

(2) Select the TV camera filter, adjust the grain, and capture the image.

(3) The captured image can be processed as is, or it can be recorded on a floppy disk and loaded into the computer 20 as necessary for image processing and calculation processing.

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

■ Input the signal from the TV camera into the image memory.

■ When the plate is set in the fixed position on the plate holder, the required location for reading and writing is automatically determined, so determine the address in memory of this location, and within that address, calculate the number of pixels with a brightness of 0 to 8 in the area. Let it be calculated as

Another method is as follows: (1) Overlaying the image input into the image memory with a mask image for erasing images other than the areas that need to be read to create an image of only the necessary areas.

■ Convert the required brightness 0 to 8 in the remaining image to F, and
Convert F to O.

■ Superimpose the image for specifying the order of image calculation and the image obtained in ■, and calculate the area of each agglomeration/9 turns.

The area of the agglutination pattern obtained by the above image processing is compared with a reference value to determine whether the agglutination reaction is positive or negative.

FIGS. 3 and 4 are schematic diagrams showing other examples in which the configuration of the 7-card is changed.

FIG. 3 shows an example in which the hood 12 is formed in a linear shape so that the light emitted from the microplate 6 directly enters the TV camera 13. FIG. 4 shows an example in which the hood 12 is bent once into an approximately rLJ shape, and the light emitted from the microplate 6 is reflected once by the mirror 14 and then enters the TV camera 13. It is.

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

FIG. 5 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 due to a change in particle concentration. When transmitted light is used in this way, in a sample with precipitates, the area where changes in particle concentration cannot be measured expands, leading to errors in aggregation determination.

On the other hand, FIG. 6 shows the measurement results of the relationship between particle concentration and brightness when reflected light was measured. In this measurement of reflected light, samples without white turbidity are expressed by ABC as in Figure 5, 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 due to cloudiness does not increase with respect to the change in particle concentration.

Measurement using the apparatus shown in Fig. 1 consists of measurement using reflected light from the main light source 8 and measurement using transmitted light from the auxiliary light source 9, so the apparatus shown in Fig. 1 includes the addition of Figs. 5 and 6. This is the measurement of Therefore, the influence of cloudiness is offset by the large cloth and is virtually eliminated.

This makes it possible to make more accurate judgments on turbid samples than when using only transmitted light.

Next, measurement results comparing the contrast between the case of only the reflected light from the main light source 8 and the case of both the reflected light and transmitted light from the main light source 8 and the auxiliary light source 9 will be described. FIG. 7 shows the results of this measurement. A in the figure represents the observed intensity of light in the case of reflected light, and in this case, for example, the contrast at a location where the transmittance is 0.6 is the magnitude shown in the figure. The opening in the figure shows the observed light intensity when both reflected light and transmitted light are used, and the contrast at the point where the transmittance is 0.6 is the size shown in B in the figure. Become. Therefore,
It can be seen that the contrast (B) in the case of both reflected light and transmitted light appears to have a stronger contrast than the contrast A in the case of only reflected light. That is, this increase in contrast is due to an increase in the amount of transmitted light, and an increased contrast corresponding to the increased amount.

〔Effect of the invention〕

Since the present invention is configured as described above and captures the optical state of the TV left camera well, it is possible to capture data from all wells of one microplate in an extremely short period of time, for example, π seconds, and a large amount of data can be captured. samples can be processed quickly. In addition, since the optical states of all wells are acquired at once, a plate moving device is not required and the operation can be simplified. moreover,
It is possible to simultaneously obtain information on the two-dimensional spread of particle aggregation, as well as information on the particle concentration in the well as a photometer. Data can be obtained at high speed.

In addition, the present invention provides light sources above and below the grating table, and both the reflected light from the upper light source and the transmitted light from the lower light source are captured by the TV left camera to determine agglomeration. It is possible to eliminate the clouding effect that occurs when using the transmitted light from the light source below, and to reduce the brightness inside the well, which was limited when using the reflected light from the light source above. You can increase the brightness and contrast by increasing the brightness. Therefore, particle aggregation can be determined extremely accurately.

[BRIEF DESCRIPTION OF THE DRAWINGS] Fig. 1 is a schematic diagram of an embodiment of the particle aggregation determination device of the present invention, Fig. 2 is a partial cross-sectional view of the same plate holder, and Figs. 3 and 4 are other parts of the image capturing device. Partial schematic diagram showing an example of
The figure shows the relationship between particle concentration and brightness when measuring transmitted light, Figure 6 shows the relationship between particle concentration and brightness when measuring reflected light, and Figure 7 shows the contrast measurement. It is a figure showing a result. l...image capture device, 2...image processing device, 5.
...Plate stand, 6...Micro grate, 7...
・Light transparent part, 8... Main light source, 9... Auxiliary light source, 1
0...Light amount adjustment plate, 12...Hood, 13...T
V left camera 14...Mirror, 17...Video input section, 1
8...Part of digital image memory, 19...Video output unit, 20...Computer, 22...Printer.

Claims (1)

[Claims] A light-transmissive plate holder on which a microplate is placed, a light source placed above and below the plate holder, and a light source arranged so that light emitted from the upper side of the microplate placed on the plate holder is incident. A particle aggregation determination device comprising: an image capture device having a TV camera; and an image processing device that processes an image signal input from the TV camera to determine an aggregation state.