JPH03191848A - Method for judging particle aggregation pattern - Google Patents

Abstract

PURPOSE:To make it possible to perform accurate judgment by picking up the image of particle aggregation pattern in two dimensions, and judging the aggregated state and non-aggregated state based on the distributing state of the pattern. CONSTITUTION:A reaction container has the slant bottom surface at at least one part. Many conical wells as the reaction containers are formed in a matrix pattern in a microplate 1. A sample and a reagent are put in the microplate, and particle aggregation reaction is performed. The formed particle aggregation pattern is judged, and the aggregation reaction is measured. A lamp 2 as a light source is arranged at the lower side of the plate 1, and a TV camera 3 as an image sensor is arranged at the upper side. The image of the particle aggregation reaction pattern is picked up in two dimensions. The image data are amplified in a signal processing circuit 4 and converted into the digital signal. Then the signal is stored in a memory 5. The pattern is read out and displayed on the screen of a monitor 6. The differentiated value is obtained in a differentiating circuit 7 at the same time. The pattern is judged based on the distribution of the differentiated value in a judging circuit 8.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention involves placing a sample and a reagent in a reaction container having at least a portion of an inclined bottom surface, causing an agglutination reaction to occur; The present invention relates to a method for determining a particle aggregation pattern and measuring an agglutination reaction.

[Conventional technology]

Using a reaction container with a conical bottom or a microplate with a large number of wells with a conical bottom,
A technique for determining the presence or absence of aggregation by determining the particle aggregation pattern formed on the bottom surface is known, and is disclosed, for example, in US Pat. No. 4,727,033. This known particle aggregation pattern determination method uses special light-receiving elements to receive light at the center and periphery of a conical bottom, and determines aggregation or non-aggregation from the ratio of the outputs of these light-receiving elements. . In other words, in the case of agglomeration, the agglomerated particles are deposited uniformly on the bottom surface, so the ratio of the output of the light-receiving element is large between the central part and the peripheral part, but in the case of non-aggregation, many particles roll on the inclined bottom surface. Since the light falls and gathers in the center, there is a large difference in the output of the light receiving element between the center and the periphery.
The output of these light receiving elements becomes a small value. In this way, aggregation or non-aggregation is determined from the ratio of the outputs from the light-receiving elements in the central part and the peripheral part. In addition, in such conventional determination methods, the value of the ratio is compared with upper and lower limit values, and when it is larger than the upper limit value, it is judged as agglomeration, and when it is smaller than the lower limit value, it is judged that there is no aggregation. When the value is between the limit values of , it is determined that the determination is impossible or unknown.

Further developing this conventional example, a photodetector scans a straight line passing through the center of the well to obtain a one-dimensional image signal, and this image signal is processed to determine the difference between the center and the periphery. has also been proposed.

In addition, Japanese Patent Application Laid-Open No. 63-58237 discloses that an image of the bottom surface of a well is taken with a television camera, the obtained image signal is processed to determine the center position of the well, and a circle with this center position as the midpoint is created. The contour of the agglomerated pattern is determined by determining the brightness difference of the image signal along the circumference, the area within the contour thus determined is determined, and this is compared with a predetermined reference value, and if it is larger than the reference value, A method is disclosed in which it is determined that the amount is agglomerated, and when it is small, it is determined that it is not agglomerated. That is,
This conventional method attempts to determine aggregation and non-aggregation by the number of particles collected in the center of the well.

In addition, Japanese Patent Application Laid-Open No. 63-256839 discloses that the center position of the well is determined in the same manner as the above method, and the agglomeration is determined based on the correlation between the size of the central part where particles gather and the standard deviation of the differential coefficient of the pattern. A method for determining non-aggregation is disclosed.

[Problems to be Solved by the Invention] The conventional method for determining particle aggregation patterns described above has the drawback that aggregation and non-aggregation cannot be clearly determined. That is, in a method in which light is received at the center and the periphery using special light-receiving elements, and agglutination or non-aggregation is determined based on the ratio of the outputs of these light-receiving elements, a large number of specimens are determined to be unknown.

Generally, the aggregation reaction is subtle, and a particle aggregation pattern with a clear outline is rarely formed, so a relatively large number of particles that are judged to be unknown appear. Patterns that are determined to be unknown in this way are visually observed by operators to determine whether they are agglutinated or non-agglutinated, but as the number of patterns that are determined to be unknown increases, the number of samples that need to be visually observed increases, making processing difficult. There is a disadvantage that efficiency is reduced, and human error is also introduced, resulting in a disadvantage that analysis accuracy and reliability are impaired. Furthermore, although the method described in JP-A No. 63-58237 can accurately determine the center of a pattern, the size of the agglomerated area formed by particles gathering at the center of the pattern is Accurate determination is difficult because it is not determined only by non-aggregation, but also changes depending on the amount of reagent or sample dispensed. Furthermore, JP-A No. 63-256839
The method described in the above publication has the disadvantage that accurate determination cannot be made because it is easily affected by bubbles, collapse, and curling contained in the particle aggregation pattern.

The purpose of the present invention is to eliminate the above-mentioned conventional drawbacks, and to be able to accurately determine agglomeration or non-aggregation without being affected by shape abnormalities such as collapse and curling, or bubbles included in particle aggregation patterns. As a result, the present invention aims to provide a particle aggregation pattern determination method that can reduce the number of specimens that require visual observation and perform analysis efficiently.

[Means and effects for solving the problem] The method for determining a particle aggregation pattern of the present invention involves placing a sample and a reagent in a reaction container having at least a portion of an inclined bottom surface, allowing a particle agglutination reaction to take place; In determining the particle aggregation pattern formed on the inclined bottom surface and measuring the aggregation reaction,
A process of two-dimensionally imaging the particle aggregation pattern formed on the bottom of the reaction vessel to obtain two-dimensional image information, a process of differentiating this two-dimensional image information to obtain a differential value, and a process of determining the distribution of this differential value. and determining a particle aggregation pattern based on the method.

In the particle aggregation pattern determination method of the present invention, when a particle aggregation pattern contains air bubbles, the image density is high at the outline of the air bubbles, and the image density is low at the collapsed or curled areas, and the image density below the bubbles is high. This was based on the fact that these shape abnormalities can be determined from the distribution state of the differential values of the two-dimensional image signal. Therefore, according to the method of the present invention, it is possible to accurately determine the particle aggregation pattern without being affected by bubbles, collapse, curling, etc., and therefore, it is possible to accurately determine the particle aggregation pattern for samples that cannot be determined and require visual re-examination. The number is reduced, and analysis efficiency can be improved. Furthermore, compared to conventional determination methods, since the method does not determine whether particles are agglomerated or non-agglutinated based only on the area of the agglomerated part, there is no error in determination due to fluctuations in the distribution of the sample or reagent.

[Example] FIG. 1 is a diagram showing the overall configuration of an example of an apparatus for implementing the particle aggregation pattern determination method of the present invention. In this example, a microplate 1 in which a large number of wells are formed in a matrix is used as a reaction container. The bottom of each well is conical. Further, the microbrake 1 is made of a transparent material such as acrylic resin. micro play
The lens 1 is placed on a drive table (not shown) and is displaced relative to the imaging optical axis. A light source 2 is disposed below the microplate 1, and an imaging device 3 capable of two-dimensionally capturing an image of a particle aggregation pattern formed on the bottom surface of the well is disposed above. In this example, the imaging device 3 includes a CCD television camera. The two-dimensional image information output from the imaging device 3 is supplied to the signal processing circuit 4, amplified by U7, and converted into a digital signal, and then supplied to the memory 5 and stored therein. The image information stored in the memory 5 is read out and supplied to the monitor 6 so that the particle aggregation pattern formed on the bottom surface of the well of the microplate 1 can be displayed on the screen. In the present invention, the two-dimensional image information read from the memory 5 is supplied to the differentiation circuit 7 to obtain a differential value. This differentiation operation is performed using a differentiation operator as described below. Figures 2 and 3 show the differential operator,
The differential operator in Figure 2 detects horizontal brightness changes (
~, The differential operator shown in FIG. 3 detects luminance changes in the vertical direction. As shown in Figure 4 and 6, a pixel area having a size of 3 x 3 is extracted, and the brightness value of each pixel is multiplied by the coefficient of the differential operator based on the calculation formula shown below and added. and Y2 are determined, and the average value of these sums is determined, and this value is used to determine the differential value Y of the pixel at the center of the pixel area.

Yl=(-1X80 (OXB) + (
IXC)+-(-2XD+(OXE)+(
2XF ) + (-iXG + (Ox)I) + (lxl
)Y 2 = (lXA 10(-2Xf+) +-
(-1XC) + (OXD + (OXE ”) +
(OXF')+(IXG)+(2XI+)+(IXT
)Y=Y1+Y2/2 The above calculation is performed while sequentially shifting the pixel areas to find the differential value over the entire surface of the two-dimensional image.

The differential value of the two-dimensional image obtained in this way is determined by the judgment circuit 8.
and sequentially store it in the memory provided there. In the present invention, the determination circuit 8 determines the particle aggregation pattern from the distribution of the differential values obtained as described above, and an example of the determination criteria will be explained with reference to the flowchart shown in FIG. .

In the present invention, a differential value is obtained for a particle aggregation pattern formed on the bottom surface of one well, and the particle aggregation pattern is determined from the distribution.

(1) 2500 pixels have a collapse differential value of 20 or more and 40 or less
If there are more than 1, it is determined that the particle aggregation pattern is disrupted. In other words, we focused on the fact that when a particle agglomeration pattern has a large collapse similar to turning over, a large number of moderate changes in shading appear, and we counted the number of pixels with a differential value of 20 to 40, and if the number was 2500 or more, It is determined that there is a collapse in the particle aggregation pattern. Since such collapse occurs in the case of an aggregation reaction, if it is determined that there is collapse, it is determined that there is aggregation, and in this case, it is determined that it is an aggregation reaction.

(2) When there are more than 10 pixels with a bubble inclusion differential value of 200 or more, it is determined that bubbles are included in the particle aggregation pattern. In other words, when there are bubbles in the particle aggregation pattern, the difference in density becomes extremely large, and therefore many large differential values appear. It is determined that it is mixed.

(3) Agglomerated (positive) The number of pixels A with a differential value of 20 or more and 50 or less is
Ratio R = A/B divided by the number of pixels B that is greater than or equal to 00 and less than or equal to 200
When the value is 22 or less, it is determined that the particles are agglomerated.

(4) No agglutination (negative) When the value of the ratio R is 40 or more, it is determined that there is no agglutination.

(5) Impossible to judge When the value of the ratio R is larger than 22 and smaller than 40, it is judged that the substance is neither agglomerated nor non-agglomerated, and it is judged that it cannot be judged. For samples determined to be undeterminable in this way, the particle aggregation pattern is visually observed or re-examined.

In the above-described determination, if it is determined that bubbles are mixed in, C is calculated by multiplying the number of pixels with a differential value of 20 to 50 by 1.2, and 0. Find the ratio E = C / D with the value multiplied by 5, and make a judgment using this ratio as the value of the ratio R of the above-mentioned judgment criteria (3) to (5),
Determine agglutination, non-aggregation, and indeterminable. in this way,
Multiply the number of pixels with a differential value of 20 to 50 by 1.2,
By multiplying the number of pixels with a differential value of 100 to 200 by 0.5, small changes in shading are emphasized and large changes in shading are reduced, thereby reducing the effect of bubbles. .

The determination result obtained in this manner is supplied to an output device 9 such as a printer and displayed.

Next, for various samples, the differential values of the image signals obtained by two-dimensionally imaging the particle aggregation patterns were obtained, and the distribution thereof was investigated. The results are shown in the table below.

The table above includes not only the number of pixels belonging to each differential value range, but also the number of pixels with a differential value greater than 200, the number of pixels between 100 and 200, the number of pixels between 20 and 40, the number of pixels between 20 and 50, and the ratio R1225. The number of pixels, the values of C and D, the value of the corrected ratio E, and the determination results are also shown below. Samples 1.6 and 7.8 are typical negative patterns, and samples 13-18 are typical positive patterns.

In addition, samples 9 to 12 were all determined to be undeterminable. Furthermore, samples 3 and 4 were determined to have air bubbles, and were further determined to be negative and positive, respectively, based on the corrected ratio E values. Sample 5 was determined to have collapsed and was ultimately determined to be positive. As described above, according to the method of the present invention, it is possible to accurately determine whether agglomeration or non-aggregation occurs even when air bubbles are mixed in or there is collapse, and even when it is determined that it cannot be determined, visual inspection or re-examination is required. The number of samples to be passed through is reduced, and processing capacity can be improved.

(Effects of the Invention) As described above, according to the determination method of the present invention, the particle pattern formed on the inclined bottom of the reaction vessel is imaged two-dimensionally, the obtained image signal is differentiated, and the differential value is calculated. Since the distribution of particles is determined, and aggregation or non-aggregation is determined from this distribution state, it is possible to accurately determine agglomeration or non-aggregation even when air bubbles are mixed into the particle aggregation pattern or the aggregation pattern is disrupted. can do. In particular, when air bubbles are mixed into the particle aggregation pattern, aggregation or non-aggregation is determined based on the corrected value, which reduces the number of samples that are judged to be undeterminable, and therefore requires visual inspection or re-inspection. This reduces the number of samples that need to be processed and increases processing power.

[Brief explanation of drawings]

FIG. 1 is a block diagram showing the configuration of an apparatus for implementing the particle aggregation pattern determination method according to the present invention, and FIGS.
FIG. 4 is a diagram showing the operator responsible for differentiating the image signal, FIG. 4 is a diagram showing the pixel area when performing differentiation, and FIG. 5 is a diagram showing a flowchart of determination. 1... Microplate 4... Signal processing circuit 6... Monitor 8... Judgment circuit 3... Television camera 5... Memory 7... Differentiating circuit 9... Output circuit

Claims (1)

[Claims] 1. A sample and a reagent are put into a reaction container having at least a portion of an inclined bottom surface to perform an agglutination reaction, and the agglutination reaction is performed by determining a particle aggregation pattern formed on the inclined bottom surface of the reaction container. In measuring, the step of two-dimensionally imaging the particle aggregation pattern formed on the inclined bottom surface of the reaction vessel to obtain two-dimensional image information, and the step of differentiating this two-dimensional image information to obtain a differential value. A method for determining a particle aggregation pattern, comprising: determining a particle aggregation pattern based on the distribution of the differential values.