JPH0145862B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for determining agglutination patterns by immunological agglutination reactions, and in particular, particle agglutination for determining various blood types and detecting antibodies and antigens from agglutination patterns of blood cell particles. This invention relates to a pattern determination method.

For example, as a method for determining blood type,
Publication No. 16798 discloses that a reaction container whose bottom surface is curved in the shape of a wine cup is used, and a 2 to 5% suspension of test blood cells obtained by centrifugation and a specific antiserum are quantitatively dispensed into the container. After stirring both, let stand,
Next, centrifugation is performed, and the reaction vessel is violently vibrated to shake out the precipitated blood cells, and then vibrated relatively slowly to quickly collect the coagulated and bonded particles in the center of the vessel, while unbound particles A method has been proposed in which the particles are redispersed in a solution to form an agglomerated pattern and this is detected photometrically.
FIG. 1 shows the configuration of a photometric detection device for agglutination patterns in such a blood type determination method. In this photometric detection device, light from a light source 1 is made to enter from above a wine cup-shaped reaction container 5 through a frosted glass 2, a diaphragm plate 3, and a lens 4, and a center opening 6a surrounding the central opening 6a is formed below the reaction container 5. A mask 6 having an annular aperture 6b is arranged, and light enters the first light receiving element 7 through the central aperture 6a, and enters the second light receiving element 10 through the optical lenses 8 and 9 passing through the annular aperture 6b. It is configured so that it is incident. According to this photometric detection device, the amount of light that passes through the center of the reaction liquid 11 in the reaction container 5 and enters the first light-receiving element 7 represents the turbidity of the center of the reaction liquid 11. The amount of light that passes through the periphery of the liquid 11 and enters the second light receiving element 10 represents the turbidity of the periphery of the reaction liquid 11, so the amount of light that passes through the center of the reaction liquid 11 is the standard. If the amount of light passing through the periphery increases compared to the standard value at the same time as the amount of light decreases compared to the standard value, this is considered to be "agglomeration".
If the amount of light passing through the center and periphery does not change with respect to the standard value, it is determined that there is no aggregation.
It can be determined that

However, in the above blood type determination method, the centrifuged reaction container 5 is shaken vigorously to separate the precipitated blood cells from the bottom of the container, so it is not effective in determining ABO blood type, which has a strong coagulant bond. Even if it is used, in the case of immunological agglutination reactions with weak binding strength, such as when determining the Rh type blood type or when detecting various irregular antibodies, antigens, _HBs antigen, etc., the reaction vessel is By vibrating 5, particles such as blood cells that are once bound separate, and a clear aggregation pattern cannot be formed, so there is a problem that the method cannot be used. In order to solve this problem, the applicant proposed in Japanese Patent Application Laid-open No. 146044/1987 that not only blood types based on natural antibodies with strong aggregation bonding force but also blood types based on irregular antibodies with extremely weak aggregation bonding force can be determined. We proposed a method for determining blood type. Such a blood type determination method uses, for example, a reaction container with a conical bottom, and contains blood corpuscles and a standard antiserum reagent in which the blood type is to be determined, and stirs them for a relatively short period of time ( Blood type is determined by detecting the agglutination pattern after allowing the device to stand for approximately 30 minutes. In this method, when the blood cell particles to be tested react with the antiserum reagent, the precipitated blood cell particles aggregate and bond, and are deposited thinly like snow on the conical bottom to form a uniform deposition pattern. If the blood cells do not react with the antiserum reagent, the blood particles do not aggregate and settle in a discrete manner. When they reach the conical bottom, they roll down the slope and gather at the center of the conical bottom to form an accumulation pattern. . Therefore, the blood type can be determined by photoelectrically detecting the difference in the pattern of precipitated blood cells depending on the presence or absence of reaction with the antiserum reagent formed on the bottom of the cone. FIG. 2 shows the configuration of a photometric detection device for agglutination patterns in this blood type determination method. Light source lamp 2
The light emitted from 1 is made into a parallel beam by a collimator lens 22, and is irradiated onto a reaction vessel 23 having a conical bottom. This light flux is modulated by the sedimentation pattern of blood cells present at the bottom of the reaction vessel 23. An image of this bottom surface is formed on a light receiving device 25 by an objective lens 24. As shown in the plan view, this light receiving device 25 consists of a light receiving element 26 that receives an image at the center of a conical bottom and a ring-shaped light receiving element 27 that receives an image at the periphery. Dynamic amplifier 28 is supplied. When blood cells are coagulated and bonded, blood cells are uniformly buried on the bottom surface, and weak light enters the light-receiving elements 26 and 27, which is somewhat blocked by the blood cell accumulation layer. The amplitudes of the output signals will be medium and equal in magnitude, and the output of the differential amplifier 28 will be at a low level. On the other hand, when blood cells are not combined, a pattern is formed on the bottom surface in which blood cell particles gather at the center, so almost no light enters the light receiving element 26, and the amplitude of the output signal is Although small, strong light enters the light receiving element 27, the amplitude of its output signal becomes large, and the output of the differential amplifier 28 becomes high level. Therefore, by comparing the output signal of the differential amplifier 28 with a predetermined reference value in the determination circuit 29, it is possible to determine whether "aggregation" or "non-aggregation" occurs, and this determination result is sent to the printer 30. It can be printed out.

However, even in such a blood type determination method, it may be difficult to determine the agglutination pattern depending on the form or shape of the agglutination pattern. In other words, the agglomeration pattern is formed in various forms during the agglomeration process.
It moves in the direction of agglomeration with a change in shape,
The final agglomerated state does not necessarily exhibit a uniform agglomerated morphology. The reason for this is that after dispensing a fixed amount of the suspension of the analyte and antiserum into a reaction container and stirring, the agglutinated components are finally collected on the bottom of the container and the agglutination pattern is determined. However, depending on the ingredients, agglomeration may often occur. Especially in the case of back-up testing of ABO blood test method,
It has been experimentally found that depending on the subject, the peripheral part of the agglutination pattern may turn over or fall off, making it difficult to determine the blood type.
It is assumed that these phenomena are caused by the subject itself, the container itself, the antiserum, the mechanism of the subject and the antiserum, and the effects of relative phenomena. However, even with the current state of technology, it is not possible to elucidate the above phenomenon, and even if there is a theory to elucidate it, it remains no more than experimental speculation. Furthermore, when performing the R-PHA method to test for _HBs antigens and the T-PHA method to test for syphilis antibodies, the aggregation binding pattern is different from the surrounding area because the _HBs antigens and syphilis antibodies are in trace amounts and have weak aggregation power. It crumbles easily and the pattern is often unclear.

When agglomeration collapse occurs in this way, even if it is originally "agglomeration", a relatively large difference occurs in the outputs of the light receiving elements 26 and 27 in FIG. 2 due to the distortion of the pattern at the periphery. In some cases, an incorrect determination result may be output, or it may be determined that "determination is not possible."

On the other hand, as a conventional particle aggregation pattern determination device, there is one disclosed in Japanese Patent Publication No. 13907/1983. FIG. 3 is a perspective view showing the configuration of a main part of a photometric detection device in such a particle aggregation pattern determining device. This photometric detection device intermittently transports a microplate 31 made of a transparent member in which a large number of reaction depressions 30 are formed in a matrix, for example, in the X-axis direction, and also in the Y-axis direction perpendicular to the direction of transport of the microcomplate 31 The aggregation pattern formed on the bottom surface of the reaction cavity 30 is detected by intermittently moving an arm 34 that integrally holds a light source 32 and three light receiving devices 33a, 33b, and 33c. The detection of the agglomeration pattern is carried out using three reaction depressions corresponding to the three light receiving devices 33a to 33c.
Detection is performed in the same manner as a block, and the arm 34 is intermittently moved in the Y-axis direction for each block.

However, even in such a photometric detection device, if the above-mentioned agglomeration failure occurs, an erroneous determination occurs, and the microplate 31 and arm 34 are Since it is difficult to transport in the axial direction and the Y-axis direction, it is not possible to adequately respond to various forms of agglomeration patterns, resulting in a problem of lowering measurement accuracy and adversely affecting normal determination results.

The purpose of the present invention is to solve the various problems mentioned above, and to stabilize the aggregation pattern caused by the immunological agglutination reaction caused by microscopic particles agglomerated, regardless of the misalignment of the scanning mechanism or the strength of the aggregation bond. Moreover, the present invention provides a particle aggregation pattern determination method that can accurately determine a particle aggregation pattern.

The present invention provides a method for photoelectrically detecting and determining a particle aggregation pattern formed on the bottom surface by precipitation of particles in a reaction solution contained in a reaction container with at least a portion of the bottom surface being inclined. illuminate,
This image of the bottom surface is scanned by a light receiving device having concentric first and second light receiving parts, and a plurality of data are acquired for each light receiving part, and a plurality of data of the first light receiving part located at the center is used to scan the image. After determining the data pattern, the maximum value or minimum value of each of the plurality of data of the first light receiving section and the plurality of data of the second light receiving section is detected based on the judgment result, and then the detected After determining whether each maximum value or minimum value is within a predetermined range, a judgment function is calculated based on each maximum value or minimum value within the predetermined range, and the judgment function and the reference number are calculated. The feature is that the agglomeration pattern is determined by comparing the two.

The present invention will be described in detail below with reference to the drawings.

FIG. 4 is a perspective view showing the configuration of an example of a particle aggregation pattern determining apparatus for implementing the method of the present invention. In this example, a reaction vessel 40 whose bottom surface is inclined in a conical shape
A microplate 41 made of a transparent member having a large number of particles arranged in a matrix on a substrate is used, and each reaction vessel 40 of this microplate 42 contains a test liquid containing particles to perform an agglutination reaction. In this example, the microplate 41 has a diameter of 10 mm in the X-axis direction.
There are 12 reaction vessels in total, 12 in the Y-axis direction perpendicular to the X-axis, and 12 reaction vessels in the Y-axis direction.
0 contains the same specimen. Therefore, one microplate 41 is used to analyze these 10 samples and 12 channels. This microplate 41 is intermittently transferred in the X-axis direction according to the arrangement pitch of the reaction vessels 40 in the X-axis direction by a transfer means (not shown). At a predetermined stopping position of the microplate 41, there is an arm 42 that can reciprocate in the Y-axis direction.
A light emitting part 43 and a light receiving part 44 are held together while facing each other so as to sandwich the microplate 41 between them, and this arm 42 is alternately moved forward and backward in synchronization with the intermittent movement of the microplate 41 in the X-axis direction. 5 is a diagram showing the structure of the light projecting section 43 and the light receiving section 44 shown in FIG. 4. The light projecting section 43 uses the light from the light source lamp 45 to pass through a heat absorption filter 46,
The configuration is such that the bottom surface of the reaction container 40 of the microplate 41 is uniformly illuminated through a condenser lens 47 and an illumination lens 48, and the light receiving section 44 transmits an image of the uniformly illuminated bottom surface of the reaction container 40 to an imaging lens 49.
The configuration is such that the light is received by the light receiving device 50 through the following steps. As shown in the plan view, the light receiving device 50 includes two light receiving elements 51 and 52 arranged concentrically and separately, and the photoelectric conversion outputs of these light receiving elements 51 and 52 are transmitted through amplifiers 53 and 54, respectively. After amplification, the analog switch 55 and the A/D converter 5
6 to the CPU 57.

In this example, since the bottom surface of each reaction container 40 is conical, when the test liquid is coagulated and bonded, a uniform accumulation pattern 61 is formed on the bottom surface of the reaction container 40 as shown in FIG. 6A. If there is no cohesive bond, the sixth
As shown in Figure B, an integrated pattern 62 is formed at the center of the conical bottom surface. Therefore, arm 42
When the reaction vessel 40 is scanned by moving the light emitting unit 43 and the light beam unit 44 together in the Y-axis direction, the light receiving element 51 detects the microplate in the case of the uniform estimation pattern of FIG. 6A. Since the thickness of the reactor 41 becomes thinner toward the center of the reactor 40, a photoelectric conversion output pattern is obtained in which the output increases near the center of the reaction vessel 40, as shown in FIG. 7A. In the case of the integrated pattern shown in FIG. 6B, a photoelectric conversion output pattern is obtained in which the output becomes smaller near the center of the reaction vessel 40, as shown in FIG. 7B.

The output pattern shown in FIG. 7A and B, that is, the photoelectric conversion output from the starting point 1 to the ending point N of the diameter of the reaction vessel 40 is all sampled and sent to the CPU 57.
However, in this example, in consideration of improving measurement accuracy and shortening the scanning processing time, the reaction vessel 4
An arbitrary scanning section n ₁ to n ₂ centered approximately at the center of 0 is set, and the light receiving elements 51 are placed at n positions at arbitrary equal intervals (1 μ to 100 μ) within this scanning section.
The photoelectric conversion output of the light receiving element 51 and the photoelectric conversion output of the light receiving element 52 when the light receiving element 51 is located at each of the above positions are sampled and taken into the CPU 57, respectively.
Note that the sampling number n of the photoelectric conversion outputs of the light receiving elements 51 and 52 depends on the measurement accuracy, the diameter of the reaction vessel 40, the form of the empirical agglomeration pattern, the scanning period,
This is determined in advance by considering various conditions such as reagents, etc. For example, if the diameter of the reaction container 40 is 5 mm to 10 mm.
If it is about 100 to 100, the number can be about 100 to 100.

The determination operation of this embodiment will be explained below with reference to the flowchart shown in FIG. In the following description, the photoelectric conversion output of the light receiving element 51 will be referred to as center data, and the photoelectric conversion output of the light receiving element 52 will be referred to as peripheral data.

First, after importing n pieces of center data and peripheral data, it is determined whether the pattern of the center data is "convex upward" (Figure 7A) or "convex downward" based on the center data. (Figure 7B). This judgment is n ₁
The center data A and the (n ₁ + α)th (α is n/
(any number less than or equal to 2) is compared with the n _second center data C and (n ₂ - α')
Compare the center data D of the th (α′=α or any number less than or equal to n/2), and when A<B, C<D, it is “convex upward”, and when A>B, C>D When A<B, C<D, or A>B,
When C<D, all of the following determinations are disabled (the final determination is set to "?").

When it is determined that it is "convex upward", the maximum value of the center data is next detected, and when it is determined that it is "convex downward", the minimum value of the center data is next detected.
To detect the maximum value, first, the maximum value (REmax) is detected among the n pieces of center data, and the peripheral data (RF) at the position where REmax is given is determined. If there are two or more maximum values, (n/2-1)
or the one closest to (n/2-2), (n/2+1) or (n/2+2) (if the degree of proximity is the same, the one with the younger sampling order, for example, the n/2nd and (n/2+2) )
If the th is the maximum value and is equal, set it to n/2th). In this example, take the average of REmax, RF, and the data at adjacent sampling positions on both sides (total of 3 each), use that value as photometric data, and use the obtained data as REmax in the center and RFmax in the periphery. . In addition, the detection of the minimum value is performed by detecting the minimum value (REmin) from the n center data and the peripheral data (RF) at that position, as well as detecting the peripheral data (RF) at the positions on both sides, in the same way as the above "maximum value detection". For each of the three pieces of data, the average of the center and peripheral areas is calculated and used as photometric data, and similarly, it is used as REmin and RFmin.

The photometric data detected by the above operation has unequal spans due to the difference in the light receiving areas of the light receiving elements 51 and 52. Therefore, Emax, Fmax or Emin, Fmin is multiplied by a correction constant so that both spans are equal.
seek.

Next, the photometric data Emax after the above correction process,
Fmax or Emin, L that is preset by Fmin.
Compare whether or not the Limit and H.Limit (see Figure 7 A and B) are within the predetermined range, and L.Limit<Emax, Fmax or Emin, Fmin<
When H, Limit, next move on to calculation of judgment function G, Emax,
When Fmax≧H.Limit or Emin, Fmin≦L.Limit, the final judgment result is “?”.

In this example, the determination function G uses the ratio G=F/E between the corrected center data E and peripheral data F,
Calculate this. Note that this judgment function G is F/E
In addition, F-E, logF/E, log(F-E) log(F-
QE) However, Q can also be a constant, etc.

Next, the value of the above-mentioned judgment function G is compared with preset high and low level thresholds H.Thr and L.Thr, and when G≧H.Thr, that is, “non-aggregation”, “−
When J, G≦L.Thr, that is, “agglomeration”,
When “+”, L.Thr<G<H.Thr, it is judged as “?”. Note that if the threshold values H.Thr and L.Thr are set too high, the accuracy of the judgment "+" will increase, but on the other hand, the probability that an undecidable "?" will appear will increase, and L.Thr
If the value is too low, the accuracy of the judgment "-" will increase, but the probability of an undeterminable "?" will also increase, so the judgment function G used, the reagent, the sample situation,
Set appropriately by considering the surrounding environment (temperature, humidity), judgment accuracy, etc.

As described above, in the present invention, an image of an agglomeration pattern formed on the bottom surface of a reaction vessel having at least a portion of the bottom surface as an inclined surface is captured by a light receiving device having concentric first and second light receiving sections. After scanning and capturing a plurality of data for each light receiving section, and determining the pattern of the data from the plurality of data of the first light receiving section located at the center, the data pattern of the first light receiving section is determined based on the determination result. After detecting the respective maximum or minimum values of the plurality of data and the plurality of data of the second light receiving section, and then determining whether each of the detected maximum or minimum values is within a predetermined range. , a determination function is determined based on each maximum value within a predetermined range, and the aggregation pattern is determined by comparing the determination function with a reference value, so even if aggregation failure occurs, the reaction vessel The agglomeration pattern can be determined with high accuracy no matter where the aggregation pattern is located on the bottom surface, such as when the center of the aggregation pattern is misaligned with the center of the bottom of the container or when the aggregation pattern is formed off-centered in one direction on the bottom surface. .

[Brief explanation of drawings]

FIG. 1, FIG. 2, and FIG. 3 are diagrams showing the configuration of a conventional agglomerated pattern side light detection device, respectively;
FIG. 4 and FIG. 5 are diagrams showing the configuration of an example of a particle aggregation pattern determination device that implements the method of the present invention;
FIGS. 6A and 6B are diagrams showing a uniform estimation pattern and an accumulation pattern formed on the bottom of the reaction vessel;
7A and 7B are diagrams showing the output pattern of the light receiving device shown in FIG. 5, and FIG. 8 is a flow chart for explaining the sequential operations of an example of the determination method of the present invention. 40... Reaction container, 41... Microplate, 42... Arm, 43... Light projecting section, 44...
Light receiving section, 45... Light source, 46... Heat absorption filter, 47... Condenser lens, 48... Illumination lens, 49... Imaging lens, 50... Light receiving device,
51, 52... Light receiving element, 53, 54... Amplifier, 55... Analog switch, 56... A/D
Converter, 57... CPU, 61... Uniform estimation pattern, 62... Accumulation pattern.

Claims (1)

[Claims] 1. In order to photoelectrically detect and judge a particle aggregation pattern formed on the bottom surface by precipitation of particles in a reaction solution contained in a reaction container having at least a part of the bottom surface as an inclined surface, uniformly illuminating the bottom surface of the container, This image of the bottom surface is scanned by a light receiving device having concentric first and second light receiving parts, and a plurality of data are acquired for each light receiving part, and a plurality of data of the first light receiving part located at the center is used to scan the image. After determining the data pattern, the maximum value or minimum value of each of the plurality of data of the first light receiving section and the plurality of data of the second light receiving section is detected based on the judgment result, and then the detected After determining whether each maximum value or minimum value is within a predetermined range, a judgment function is determined based on each maximum value or minimum value within the predetermined range, and the judgment function and the reference value are calculated. A method for determining a particle aggregation pattern, characterized in that the aggregation pattern is determined by comparing the two.