Method of discriminating particle aggregation pattern

The aggregation pattern of particles is determined by obtaining luminous intensity curves representative of the particle aggregation pattern, obtaining a first threshold face by cutting the curves with a plane positioned at a first given height, obtaining a second threshold face by cutting the curves with a plane positioned at a second given height and determining the particle aggregation pattern by calculating the ratio of the area of the first threshold face to the area of the second threshold face.

The present invention relates to a particle aggregation pattern 
discriminating method and, more particularly, to a particle aggregation 
pattern discriminating method suitable for use in the discrimination of 
various blood types from the aggregation reactive pattern of blood 
corpuscle particles by what is called a microtiter method, in the clinical 
laboratory and in the detection of antigen and antibody. 
In the medical field, there has widely been used a method whereby 
aggregation patterns of blood corpuscle particles, latex particles and 
carbon particles are discriminated and various components (for instance, 
blood type, various antibodies, various proteins and the like) in the 
blood, virus, and the like are detected and analyzed. The microtiter 
method is relatively frequently used as a method of discriminating the 
aggregation patterns. 
In the microtiter method of immunological measurement, there has widely 
been used a method whereby the presence or absence of aggregation of 
components on a microplate is detected and a microamount of immune 
components is measured. In most cases, the presence or absence of 
aggregation is discriminated by observation by the eyes of the analyzer. 
In observation discrimination, the presence or absence of aggregation is 
synthetically discriminated by the human eyes by recognizing a 
distribution of particles in the well (reactive vessel) as an area whose 
luminance is a certain degree or less, or by comparing such a distribution 
with a standard aggregation pattern or a standard nonaggregation pattern, 
or further by making a continuous stage dilution series of specimen 
samples, or the like. Therefore, an advanced skill is needed for 
observation discrimination and such a method is a sensory discriminating 
method. Therefore, there are inconveniences such that a personal 
difference occurs due to the person who discriminates and, further, even 
when the same person discriminates, there is a lack of reproducibility. 
Automatization of observation discrimination by an apparatus results in 
that not only is labor saved, but also the discrimination results are 
objective and an improvement in measuring accuracy can be expected. 
Therefore, hitherto, many methods of automatically discriminating particle 
aggregation patterns have been studied, developed and proposed. For 
instance, in JP-B-61-59454, there is disclosed a method whereby a 
one-dimensional photosensitive element is arranged at the center of a 
concave portion of the bottom surface of a reactive vessel of a microplate 
and an aggregation image, which is formed on the bottom surface of the 
reactive vessel, is photoelectrically detected and discriminated. In 
JP-A-59-132338, there is disclosed a method whereby a number of single 
photosensitive elements are arranged and the shape of the aggregation 
image is discriminated. In JP-A-61-215948, there is disclosed a method 
whereby an aggregation image is picked up by a television camera and the 
aggregation image is discriminated. 
However, in the invention disclosed in JP-B-61-59454, the image is 
discriminated at one cross-sectional face of the aggregation image. This 
results in a problem in that the image is discriminated at one 
cross-sectional face of the aggregation image and if the center of the 
image deviates from the center of the concave portion of the bottom 
surface of the reactive vessel or if the whole image is distorted, it is 
difficult to accurately discriminate. In addition, there is also a problem 
in that the sensor must be made coincident with the center of the concave 
portion of the reactive vessel and high mechanical accuracy is required to 
position both of them. 
On the other hand, according to the invention disclosed in JP-A-59-132338, 
there is the inconvenience of the resolution being bad (1 to 2 mm) due to 
the limitation in the shape of element and it is difficult to accurately 
discriminate the aggregation pattern. 
Further, according to the invention disclosed in JP-A-61-215948, there is 
the inconvenience of it being difficult to uniformly discriminate the 
aggregation images of the reactive vessel in the central and peripheral 
portions of a microplate. Further, there is also the inconvenience that 
the amount of data to be processed in each of the reactive vessels is 
extremely large and takes a great deal of time to discriminate. 
It is an object of the present invention to improve the inconveniences of 
the conventional methods and to provide a particle aggregation pattern 
discriminating method which can improve the discriminating accuracy and 
can discriminate at a high speed, particularly, as compared with 
conventional methods. 
Therefore, according to the present invention, there is provided a particle 
aggregation pattern discriminating method in which there is provided an 
aggregation reaction checking plate having one or more reactive vessels in 
which at least a part of a bottom surface is formed as an inclined 
surface. The bottom surface of each of the reactive vessels is uniformly 
irradiated by light emitting means arranged on one side of an aggregation 
reaction checking plate and the transmitted light is received by 
photosensing means arranged on the other side of the aggregation reaction 
checking plate through an image forming lens. Particles in a reactive 
solution enclosed in each of the reactive vessels sediment, and a particle 
aggregation pattern, formed on the bottom surface, is photoelectrically 
detected and discriminated, wherein a one-dimensional photosensitive 
element is used as the photosensing means. By moving the one-dimensional 
photosensitive element, the transmitted light is continuously received, an 
image formed on the bottom surface of each of the reactive vessels is 
retrieved as photosensitive data, an output signal of the one-dimensional 
photosensitive element is continuously processed, a number of transmitted 
luminous intensity curves are made, intersection points of a solid which 
is obtained from a number of transmitted luminous intensity curves and 
preset threshold faces are obtained. Two points on each of the transmitted 
luminous intensity curves having a predetermined relationship around each 
of the intersection points as a center are calculated, first and second 
pseudo faces are obtained by sequentially connecting those points, and the 
particle aggregation pattern is discriminated by the area ratio of the 
first and second pseudo faces. Due to this, the above object of the 
present invention is accomplished.

Embodiments of the present invention will be described hereinbelow on the 
basis of FIGS. 1 to 11. 
FIG. 1 shows an example of an apparatus which embodies a particle 
aggregation pattern discriminating method according to the present 
invention. 
An aggregation reaction detecting apparatus 20 shown in FIG. 1 comprises a 
horizontal plate 11, a supporting member 12A and another supporting member 
12B for supporting the horizontal plate 11 at a bottom surface thereof. An 
opening 11A is formed in a part of the horizontal plate 11. A microplate 
1, as an aggregation reaction checking plate, is attached to this opening. 
As shown in FIG. 3, the microplate 1 comprises a translucent board 1b in 
which a number of reactive vessels la, each of which having its bottom 
surface formed like a cone, are arranged and formed in a matrix. In this 
embodiment, as the microplate 1, there is used a microplate in which the 
reactive vessels 1a are arranged and formed in a matrix of eight rows and 
twelve columns. 
A reinforcing plate 12C, which couples and fixes both supporting members 
12A and 12B, is attached therebetween. On the other hand, as shown in FIG. 
2, a guide shaft 13 is attached between the supporting members 12A and 12B 
along the longitudinal direction of the horizontal plate 11. Further, 
another shaft 14, in which a male screw of a ball screw is formed along 
the whole length, is arranged between the supporting members 12A and 12B 
in parallel with the guide shaft 13 and is rotatably installed. 
A box 15, shown in FIGS. 1 and 2, is attached to both of the shafts 13 and 
14 so that it can reciprocate along both shafts 13 and 14. Practically 
speaking, a hole 15a having a diameter almost equal to the diameter of 
shaft 13 and a hole 15b having a diameter almost equal to the diameter of 
shaft 14 are formed in the box 15. On the other hand, a female screw 
portion of the ball screw, in which a female screw (not shown) is formed 
and which faces the foregoing male screw through a ball (not shown), is 
provided in the box 15. 
A movable plate 16, on which is mounted a photosensitive unit 10 shown in 
FIGS. 3 and 4, is arranged and fixed onto the upper surface of the box 15 
in parallel with the horizontal plate 11. Supporting plates 18A and 18B 
for supporting both ends of an upper plate 17, in which light emitting 
diodes 2A shown in FIG. 3 are fixed to the lower surface thereof, are 
fixed onto the upper surface of the movable plate 16 so as to 
perpendicularly cross the movable plate 16. Light diffusing plates 31 and 
32 shown in FIG. 3 are integrally held to the lower surface of the upper 
plate 17. On the other hand, an LED driver circuit 8 for driving light 
emitting diodes 2A comprising ICs, or the like, is provided under the 
lower surface of the upper plate 17 (refer to FIG. 3). 
A board 19 arranged in parallel with the movable plate 16 is fixed onto the 
upper surface of the movable plate 16. 
A CCD driver circuit 9 for driving a one-dimensional CCD sensor 3A, which 
comprises an IC or the like and will be explained hereafter, is attached 
to the board 19. 
Further, the two photosensitive units 10, constructed as shown in FIG. 3, 
are arranged on the upper surface of the movable plate 16 in a manner such 
that a part in the longitudinal direction of each photosensitive unit 10 
mutually overlaps. The photosensitive units 10 are arranged along the 
vertical columns of the reactive vessels 1a, which are arranged in a 
matrix on the microplate 1. The photosensitive units 10 are coupled by a 
coupling member 10A as shown in FIG. 4. 
As shown in FIG. 3, the photosensitive unit 10 comprises: a lens holder 5; 
image forming lenses 4 held to the lens holder 5; and a one-dimensional 
CCD sensor 3A as a one-dimensional photosensitive element which is 
attached at the bottom portion of the lens holder 5. 
Explanation will be made in further detail. A plurality of holes (in the 
embodiment, four holes) 5a are formed in the lens holder 5 at intervals 
equal to the distances among the reactive vessels la which are neighboring 
along the longitudinal direction. Each of the image forming lenses 4 is 
fixed to a peripheral wall portion of each hole 5a. The one-dimensional 
CCD sensor 3A is positioned at the bottom portion of the lens holder 5 in 
parallel with the microplate 1 so as to be spaced downwardly from the 
image forming lens 4 by a predetermined distance, that is, by almost the 
same distance as the focal distance of the image forming lens 4. 
The photosensitive units 10 are fixed onto the upper surface of the movable 
plate 16 in such a manner that the four holes 5a formed at intervals equal 
to the distance between the reactive vessels 1a, which are adjacent in the 
longitudinal direction, coincide with the reactive vessels 1a. 
In FIG. 3, the light emitting diodes 2A, as light emitting means, are 
arranged above the microplate 1 so as to face the image forming lenses 4. 
The two light diffusing plates 31 and 32 are arranged between the light 
emitting diodes 2A and the microplate 1 so as to be in parallel with each 
other and be spaced from each other by a predetermined interval. The light 
emitting diodes 2A and the light diffusing plates 31 and 32 are integrally 
provided on the lower surface side of the upper plate 17 together with the 
LED driver circuit 8. 
A motor 21 for applying a rotational force to shaft 14, through a gear 
mechanism (not shown), is attached to the outside of the supporting member 
12A. Therefore, in the present embodiment, when the motor 21 is driven, 
the movable plate 16 and upper plate 17 can integrally reciprocate in a 
manner such that the horizontal plate 11 and microplate 1 are sandwiched 
at their upper and lower positions and in the direction of arrow P in FIG. 
1, that is, along the lateral columns of the reactive vessels la arranged 
like a matrix on the microplate 1. 
The operation of the aggregation reaction detecting apparatus 20 
constructed as described above will now be described. 
When the motor 21 is driven, the movable plate 16 is put into motion. A 
positioning means (not shown) is controlled by a CPU (not shown). When the 
photosensitive units 10 shown in FIG. 2 are moved and set below arbitrary 
vertical columns of the reactive vessels la formed on the microplate 1, 
the lights from the light emitting diodes 2A are irradiated onto the 
microplate 1 through the light diffusing plates 31 and 32. Images of 
aggregation patterns which are formed on the bottom surfaces of the 
reactive vessels 1a, located above the photosensitive unit 10, are formed 
onto the one-dimensional CCD sensors 3A through the image forming lenses 4 
by the irradiation lights from the light emitting diodes 2A. 
Output signals from the one-dimensional CCD sensors 3A are sent to the CPU 
(not shown) through A/D converters (not shown). The CPU calculates which 
reactive vessel is being examined by obtaining a movement amount of the 
movable plate 16 from a feed amount (rotation amount) of the motor and 
automatically discriminates the aggregation patterns of the specimens in 
the reactive vessels as will be explained hereinafter. 
FIGS. 5a and 5b show typical examples of the aggregation pattern on the 
bottom surface of the reactive vessel 1a. FIG. 5a shows a collection 
pattern in the case where no aggregation coupling reaction occurs and the 
sedimented particles roll and drop into the inclined bottom surface of the 
reactive vessel 1a and are collected near the center. FIG. 5b shows a 
pattern in the case where the aggregation reaction occurs and the 
particles are uniformly deposited like a snow over the conical bottom 
surface of the reactive vessel 1a. 
FIG. 6a and 6b show transmitted luminous intensity curves which are 
obtained by processing the output signal of the one-dimensional CCD sensor 
3A when scanning the patterns shown in FIG. 5a and 5b by the CCD sensor 
3A. FIG. 6a shows the curve corresponding to the collection pattern of 
FIG. 5a. FIG. 6bshows the curve corresponding to the uniform deposition 
pattern of FIG. 5b. 
In FIG. 5a, the amount of light transmittance associated with the particles 
collected near the center of the reactive vessel is relatively low in 
comparison to the amount of light transmittance associated with the 
surrounding area where no particles are present (see also FIG. 3). The 
luminous intensity curves of FIG. 6a exhibit a marked increase in 
magnitude corresponding to the collected particles of FIG. 5a and the low 
light transmittance associated therewith. In FIG. 5b, the amount of light 
transmittance is generally uniform across the bottom surface of the vessel 
due to the uniformly deposited particles. Thus, the luminous intensity 
curves of FIG. 6b exhibit a more uniform magnitude than those of FIG. 6a. 
An aggregation pattern discriminating method of the present invention will 
now be described on the basis of FIGS. 7 to 10. 
FIGS. 7a and 7b show examples of a plurality of transmitted luminous 
intensity curves obtained in a manner similar to FIGS. 6a and 6b and the 
result of the two-dimensional data processes. 
The hatched portion in FIG. 7a is a face (threshold face) which is obtained 
by connecting the points which are obtained by cutting a plurality of 
transmitted luminous intensity curves at a predetermined threshold level 
by plane, for instance, at 3/4 of the maximum height h of the image 
obtained from each curve. As shown in FIG. 7b, assuming that lengths of 
portions where the transmitted luminous intensity curves intersect the 
above face are set to 1.sub.1, 1.sub.2, 1.sub.3, . . . and .DELTA.x = 
sampling interval, from what is called a quadrature by parts, 
EQU S=(1.sub.1 .multidot..DELTA.x+1.sub.2 .multidot..alpha.x+ . . . +1.sub.n 
.multidot..DELTA.x) 
(where, n .fwdarw. .infin.) 
is nothing but an area of the above face. 
As mentioned above, the output of the one-dimensional CCD sensor 3A is 
converted into two-dimensional data. 
FIGS. 8a and 8b are an explanatory diagram showing a first practical method 
of the aggregation pattern discriminating method of the invention. 
It is assumed that a transmitted luminous intensity curve function is f(x). 
The first discriminating method is performed according to the following 
procedure. 
1. The transmitted luminous intensity curve function f(x) is scanned. 
2. With respect to the transmitted luminous intensity curve functions 
f.sub.l (x) to f.sub.n (x) obtained by scanning, 
EQU {[f(x)].sub.max +[f(x)].sub.min }/2=h 
is obtained. The values of h determine the threshold face. 
3. The intersection points of the resultant h and the transmitted luminous 
intensity curve function f.sub.m (x) are set as the center of the check 
level. Intersection points P.sub.l and Q.sub.l and R.sub.l and T.sub.l on 
an ordinate axis of the points which are away from the check level center 
in the direction of an abscissa axis by a predetermined width L and the 
transmitted luminous intensity curve function, for instance f.sub.l (X), 
shown in FIG. 8a, are obtained. The length = 1.sub.1 of line segment 
P.sub.l Q.sub.l and the length = 1.sub.2 of line segment R.sub.l T.sub.l 
are calculated. Similarly, intersection points P.sub.k and Q.sub.k and 
R.sub.k and T.sub.k with an ordinate axis of the points which are away 
from a check level center h.sub.m by a predetermined Width L are obtained 
with respect to each of the transmitted luminous intensity curve 
functions. A length = 1.sub.2k-1 of the line segment P.sub.k Q.sub.k and a 
length = 1.sub.2k (k = 2, 3, . . . , n) of the line segment R.sub.k 
T.sub.k are calculated. 
4. Thereafter, an area S.sub.n = 1.sub.1 + 1.sub.3 + . . . + 1.sub.2n-1 of 
the first pseudo face and an area S.sub.n+1 = 1.sub.2 + 1.sub.4 + . . . + 
1.sub.2n of the second pseudo face are obtained, thereby discriminating 
the aggregation or nonaggregation by the magnitude of S.sub.n /S.sub.n+1. 
FIG. 8a shows an example of the discrimination of the nonaggregation 
pattern. FIG. 8b shows an example of the discrimination of the aggregation 
pattern. The method of FIGS. 8a and 8b is a especially effective method 
because when there is a distortion of the center or edge of the bottom 
surface of the reactive vessel of the microplate, the adverse influence of 
the distortion can be eliminated. 
FIG. 9a and 9b are explanatory diagrams showing the second practical method 
of an aggregation pattern discriminating method of the invention. 
The second discriminating method is executed in the following procedure. 
1. The transmitted luminous intensity curve function f(x) is scanned. 
2. With respect to the transmitted luminous intensity curve functions 
f.sub.l (x) to f.sub.n (x) obtained by scanning, 
EQU {[f(x)].sub.max +[f(x)].sub.min }/2=h 
is obtained. 
3. The intersection of the resultant h and a transmitted luminous intensity 
curve function f.sub.m (x) is set to a check level center. Intersection 
points P.sub.k and Q.sub.k and R.sub.k and T.sub.k (k = 1, 2, 3, . . . , 
n) on an abscissa axis of the points which are away from the check level 
center in the direction of an ordinate axis by a predetermined width L and 
the transmitted luminous intensity curve function are obtained in a manner 
similar to the above first discriminating method. The line segment P.sub.k 
Q.sub.k = 1.sub.2k-1 and the line segment R.sub.k T.sub.k = 1.sub.2k (k = 
1, 2, 3, . . . , n) are calculated. 
4. After that, an area S.sub.n = 1.sub.1 + 1.sub.3 + . . . 1.sub.2n-1 of 
the first pseudo face and an area S.sub.n+1 = 1.sub.2 + 1.sub.4 + . . . + 
1.sub.2n of the second pseudo face are obtained, thereby discriminating 
the aggregation or nonaggregation by the magnitude of S.sub.n /S.sub.n+1. 
FIG. 9a shows an example of the discrimination of the nonaggregation 
pattern. FIG. 9b shows an example of the discrimination of the aggregation 
pattern. The method of FIGS. 9a and 9b particularly is effective in the 
evaluation at a high sensitivity at a predetermined light amount width in 
the portion of a large change amount of f(x). 
FIG. 10a and 10b are explanatory diagram showing the third practical method 
of an aggregation pattern discriminating method of the invention. 
The third discriminating method will be executed by the following 
procedure. 
1. The transmitting luminous intensity curve function f(x) is scanned. 
2. With respect to the transmitted luminous intensity curve functions 
f.sub.l (x) to f.sub.n (x) obtained by scanning, the intersection points 
P.sub.k and Q.sub.k and R.sub.k and T.sub.k (k = 1, 2, 3, . . . , n) with 
first and second threshold faces h.sub.U and h.sub.L which have been 
preset by a reference aggregation pattern image are obtained as above. The 
line segment P.sub.k Q.sub.k = 1.sub.2k-1 and the line segment R.sub.k 
T.sub.k = 1.sub.2k (k = 1, 2, 3, . . . , n) are calculated. 
4. After that, S.sub.n = 1.sub.1 + 1.sub.3 + . . . + 1.sub.2n-1 and 
S.sub.n+1 = 1.sub.2 + 1.sub.4 + . . . + 1.sub.2n are obtained. By 
comparing the magnitude of S.sub.n /S.sub.n+1 with that of the reference 
aggregation pattern image, the aggregation or nonaggregation is 
discriminated. 
FIG. 10a shows an example of the discrimination of the nonaggregation 
pattern. FIG. 10b shows an example of the discrimination of the 
aggregation pattern. The method of FIGS. 10a and 10b are effective for 
discrimination at high speed because, particularly, in applications where 
a reference exists, the processes are simplest. 
FIG. 11 shows the case where in each of the aggregation and nonaggregation 
patterns of FIGS. 10a and 10b, its cross point is selected to h.sub.U. In 
this case, the discrimination is performed by the magnitude of S.sub.n+1 
'/S.sub.n+1. 
As described above, according to the invention, the one-dimensional 
photosensitive element is used as a photosensing means, by moving the 
one-dimensional photosensitive element, the transmitted light is 
continuously received, an image of which is formed on the bottom surface 
of each of the reactive vessels and retrieved as photosensitive data, an 
output signal of the one-dimensional photosensitive element is 
continuously processed, a number of transmitted luminous intensity curves 
are made, intersection points of a solid which is obtained from a number 
of transmitted luminous intensity curves and preset threshold faces are 
obtained, two points on each of the transmitted luminous intensity curves 
having a predetermined relation around each of the intersection points as 
a center are calculated, first and second pseudo faces are obtained by 
sequentially connecting those points, and an aggregation pattern is 
three-dimensionally discriminated by a method whereby the particle 
aggregation pattern is discriminated by the area ratio of the first and 
second pseudo faces or the like. Therefore, as compared with the method of 
discriminating by one-dimensional or two-dimensional data, a higher 
recognizing ratio can be obtained. The resolution can be improved to the 
resolution (.mu.m level) of the one-dimensional photosensitive element 
itself. The optimum data can be obtained by merely continuously moving the 
one-dimensional photosensitive element. Further, it is possible to 
eliminate the data of the portion which exerts an adverse influence to the 
measurement due to the distortion of the center or edge of the bottom 
portion of the reactive vessel. Thus, it is possible to provide an 
excellent particle aggregation pattern discriminating method in which the 
measuring accuracy can be remarkably improved and the high speed 
discrimination can be performed and which is not obtained hitherto.