JPH10267827A - Particle-aggregation measuring apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform measurement in a state close to that inside a living body by a method wherein blood or the like to which a fluorescent reagent is added is formed to be a slender sample flow surrounded by a sheath liquid inside a sheath flow cell and the blood is irradiated with light so as to be analyzed by detecting fluorescence or scattered light. SOLUTION: A particle-aggregation measuring apparatus which measures the ratio or the like of a platelet aggregate clot to all platelets is provided with a sheath flow cell 5, with a light irradiation means such as a continuous laser light-emitting device 21 or the like, with a detection means such as a photodiode 27 or the like, with an imaging means such as a video camera 10 or the like, with a calculation means which is constituted of a microcomputer, with a judgment means, with a discrimination means and with a computing means. Then, e.g. a whole blood sample is mixed with a fluorescent reagent and a diluent liquid, this mixture is irradiated with a laser inside the sheath flow cell 5, and the intensity of forward scattered light and the intensity of forward fluorescence are measured. They are converted into a feature parameter, and, when its value is within the region of, e.g. a platelet aggregation, its particles are photographed. On the basis of image data and the feature parameter, a platelet aggregation rate or the like is calculated and displayed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a particle aggregation measuring apparatus, and more particularly to a particle aggregation measuring apparatus for calculating a ratio of platelet aggregates to total platelets.

[0002]

2. Description of the Related Art A platelet function test has been conducted to examine a decrease or an increase in the function of platelets. Platelet function tests include various tests such as platelet adhesion function and platelet release ability, and a platelet aggregation test for checking platelet aggregation is often performed. In the platelet aggregation test, an absorbance method for measuring the degree of aggregation of platelets in platelet-rich plasma PRP (Platelet Rich Plasma) as a change in absorbance (Japanese Society of Internal Medicine, Vol. 80, No. 6, June 10, 1991) ) Is conventionally used. This absorbance method uses a fixed amount of PRP
Is used to detect the degree of light shielding (absorbance) due to platelet aggregates, and to detect relatively large aggregates (one hundred or so single platelets aggregated). I have.

[0003]

However, in a clinical setting, the so-called thrombotic preparation state before a thrombus occurs in an arteriosclerosis, hyperlipidemia, diabetes mellitus, hypertension, etc., which is one of the adult diseases, is an early stage. Hope to discover.

In the thrombus preparation state, weak aggregation of platelets (approximately several aggregations) appears as an initial stage, so that a measurement method capable of detecting weak aggregation is required. Further, detection of weak aggregation is also desired in the development of antiplatelet agents and safety tests during drug discovery. In the conventionally used absorbance method, the intensity of scattered light by each particle is measured, but it is difficult to detect weak aggregation of such particles.
In addition, since measurement cannot be performed in a whole blood state, it is not possible to measure weak aggregation in a state close to a living body.

It is known that the conventional method using PRP has the following problems. In addition to removing large platelets by the centrifugation operation performed at the time of preparing the PRP, the platelet function may be affected by the centrifugal force. In addition, it is known that blood cells other than platelets are involved in the formation of a thrombus in a living body. However, red blood cells and white blood cells are removed from PRP, so that aggregation similar to that of a living body is not reflected. In addition, the preparation of PRP requires a complicated operation such as centrifugation.

In view of the above circumstances, the present invention measures the scattered light intensity and the like and, at the same time, captures and discriminates a weak platelet aggregation particle image to determine the weak platelet aggregation number. An object of the present invention is to provide a platelet measurement device that quantitatively measures a ratio in a whole blood state close to a living body. According to the present invention, it is not necessary to prepare PRP, and measurement can be performed on whole blood, so that the platelet aggregation ability can be measured more easily and in a state closer to the inside of a living body.

[0007]

SUMMARY OF THE INVENTION The present invention provides a sheath flow cell for forming a flow of particles into a thin sample flow surrounded by a sheath liquid, light irradiation means for irradiating the sample flow with light, and an irradiated sample. Detecting means for detecting light obtained from the flow, calculating means for calculating a characteristic parameter of the particle from the light detected by the detecting means, and a target particle specified by the calculated characteristic parameter is a particle aggregation region set in advance. Determination means for determining whether or not the particles are included in the image; imaging means for capturing an image of the particles determined to be included in the particle aggregation area; and identification means for identifying the particle aggregate from the form of the captured particle image. Calculating means for calculating the particle aggregation rate from the number of particle aggregates identified by the identification means and the number of target particles included in the sample stream determined based on the characteristic parameters. There is provided a particle agglutination measurement apparatus according to claim and.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A sheath flow cell according to the present invention
This is a flow cell capable of forming a thin flow of a sample liquid by a hydrodynamic effect by wrapping a sample liquid containing particles in a sheath liquid and flowing the same, and a conventionally known flow cell can be used for the flow cell.

[0009] The sample liquid contains target particles.
Although the particles targeted by the present invention are mainly blood cells and cells contained in blood and urine, microorganisms such as yeasts and lactic acid bacteria may be measured. In order to classify the types of blood cells and cells, granules or nucleic acids in cells may be reacted with a specific fluorescent reagent in advance, and the fluorescence intensity thereof may be measured. Usable fluorescent reagents include auramine O, acridine orange, propidium iodide, ethidium bromide, Hoechst 33342, pyronin Y,
Rhodamine 123 and the like. Using such a fluorescent reagent, it is possible to suitably measure the aggregation of platelets contained in blood.

As the light irradiating means, a continuous light source such as a laser, a halogen lamp or a tungsten lamp for continuously irradiating light can be used. As the detection means, one based on electrical detection, one based on optical detection, or the like can be used. For example, light detecting means (for example, a photodiode,
A phototransistor or a photomultiplier tube) can be used.

The calculating means calculates necessary characteristic parameters such as the scattered light signal intensity, the fluorescent signal intensity, the scattered light signal pulse width and the fluorescent signal pulse width from the detected light. A video camera for capturing a two-dimensional image is generally used as a capturing unit for capturing an image of particles, but an image intensifier for amplifying a weak fluorescent image may be used. Further, the image intensifier may be provided with shutter means.

It is preferable to provide a light source for irradiating the sample stream with light for particle imaging.
A continuous light source that continuously emits light, such as a halogen lamp or a tungsten lamp, or a pulsed laser (for example,
An intermittent light source that emits light intermittently, such as Spectra-Physics, 7000 series, or a multi-strobe (for example, DSX series, manufactured by Sugawara Laboratories Inc.) can be used.

When a continuous light source is used for photographing particles, it is usually preferable to use an intermittent light source in combination with an optical shutter. As the optical shutter, a known acousto-optic effect element (acounsto-optic modulator 9)
Alternatively, an electro-optic modulator or the like can be used. This light source and the photographing means
It is preferable that light is emitted from the light source so as to be orthogonal to the sample flow, and that the imaging means is arranged on the optical axis.

The calculating means, the determining means, the identifying means and the calculating means can be constituted by a microcomputer comprising a CPU, a ROM, a RAM, a timer, an I / O interface and the like. The function of each means is such that the CPU is stored in a RAM or the like. It is executed by operating according to the procedure of the program.

Further, the identification means of the present invention includes an edge extraction means for extracting a contour of the particle image, an analysis means for obtaining an image parameter for specifying a form of the particle image based on the extracted contour, Comparing means for comparing the data with the previously set particle aggregate data, and as a result of the comparison, substantially coincident particles can be identified as particle aggregates. Here, the image parameters include, for example, the area of the particle image, the equivalent circle diameter, and the circularity.

Further, the determining means of the present invention includes a distribution map creating means for creating a distribution map corresponding to the calculated characteristic parameters, and a frequency distribution for a region containing platelets from the created distribution map, Setting means for setting the particle aggregation region based on the statistical parameter of the frequency distribution. Here, the statistical parameters are, for example, an average value and a standard deviation. The distribution diagram is a general term for a distribution of characteristic parameters, and is, for example, a two-dimensional scattergram.

Further, after the detection means is formed in the first basin of the sample flow flowing through the flow cell and the particles detected by the detection means are determined to be included in the particle aggregation region by the determination means, the particles are removed. Preferably, the imaging means is formed in a second basin of the sample stream so that imaging can be performed.

Hereinafter, the present invention will be described in detail based on an embodiment shown in the drawings. Note that the present invention is not limited to this.

FIG. 1 shows an optical system in an embodiment of the measuring apparatus according to the present invention. In this embodiment, two light sources are provided: a continuous light laser light source 21 for detecting scattered light and fluorescence, and a pulse light source 8 for capturing a particle image. Lights L1 and L2 from these two light sources 21 and 8
Irradiates the sheath flow cell 5 (in FIG. 1, the sample flow flows in a direction perpendicular to the plane of the paper) at 90 ° so as to be orthogonal to each other.

Further, in this embodiment, the pulse light for imaging the particle image is applied to the sample flow in the sheath flow cell 5 from the irradiation position of the continuous emission laser light source 21 (for example, 0 μm) as shown in FIG. Irradiation is performed on the downstream side. By shifting the irradiation position, a clear particle image that is not affected by the scattered light or the fluorescence by the cells can be taken.

The continuous emission laser light is supplied to the condenser lens 2
4 irradiates the sample flow which is narrowed down and guided to the sheath flow cell 5. When particles flow into the irradiation area, scattered light and fluorescence by the particles are collected by the condenser lens 25, and the scattered light is reflected by the dichroic mirror 26 and received by the photodiode 27. The green and red fluorescent lights are dichroic mirrors 2 respectively.
8, reflected by the mirror 29, the photomultiplier tube 30,
At 31, light is received and multiplied.

Photodiode 27, photomultiplier tube 3
The scattered light intensity signal S1 and the fluorescence intensity signals S2, S3 detected by 0, 31 are passed to a signal processing device (not shown), and information such as the height, area, and width of each detected signal pulse is A / D. Obtained as converted data.

For example, four characteristic parameters of a scattered light intensity a, a signal pulse width b, a green fluorescence intensity c, and a red fluorescence intensity d are obtained by the signal processing device. Then, the type of each particle is identified in real time using these parameters, and control is performed so that only particles of a type (for example, platelets) designated in advance as an imaging target can be selected and imaged. That is, the characteristic parameter of a certain particle is compared in real time whether or not it matches the characteristic parameter of the particle of the type to be imaged, and if it is determined that the particle is the particle to be imaged as a result, the particle is determined. A light emission trigger signal for imaging is supplied to the pulse light source 8.

The pulse light source 8 is a type of light source that emits light only momentarily (about several tens of nanoseconds) by the light emission trigger signal Ts. Even if the flow velocity of the sample flow is as high as several m / sec, the flowing light particles are shaken. An image can be taken without any. As shown in FIG. 2, the pulsed light is guided to the sheath flow cell 5 by the optical fiber 22, narrowed down by the condenser lens 23, and irradiated to the sample flow. By irradiating through the optical fiber 22, the coherency of the pulse light is reduced,
A particle image with few diffraction fringes can be captured. The pulse light transmitted through the sample flow is imaged on the light receiving surface of the video camera 10 by the projection lens 24, and a transmitted light image of the cell is captured. A video signal Vs from the video camera 10 is passed to an image processing device 11 (not shown), and is stored and stored as a digital image.

The image processing apparatus analyzes the stored particle image and extracts the edge of the particle image, the area, the equivalent circle diameter, and the like.
Image parameters such as circularity are calculated. The calculated image parameters are compared with preset data on platelet aggregation to determine whether the particle image is a platelet aggregate.

The characteristic parameters a to d, such as the scattered light intensity and the fluorescence intensity, are passed from the signal processing device to the image processing device 11 to analyze and display a scattergram (two-dimensional frequency distribution) by a combination of these parameters. Then, the aggregation rate is calculated. The above is the outline of the configuration of the platelet measurement device of the present invention.

Next, the platelet aggregate of the present invention is detected,
An example of calculating the aggregation rate of the whole platelets will be described. FIG. 3 shows a flowchart for detecting platelet aggregation according to the present invention. It mainly consists of the following four processes from S1 to S4.

Pretreatment S1: 200 μL of a whole blood sample is aspirated into the measuring apparatus of the present invention, and 10 μL thereof is used for this measurement. The sample used for measurement was immediately 40 μL of a 2.6% auramine O reagent solution (95.9%
Ethylene glycol) and 1950 μL of diluent (buffer). Due to this mixing, the platelets are fluorescently stained within a very short time.

Detection processing S2: 2.8 μL of the sample solution mixed and fluorescently stained in preprocessing S1 is irradiated with a 488 nm argon ion laser beam in a sheath flow cell. The laser light scattered by each particle in the sample liquid is photoelectrically changed by the photodiode and the photomultiplier, and the forward scattered light intensity (FSC: Forward Scatter I
ntensity) and Forward Fluorescence Intensity (FL)
sity) is measured. The electric signal subjected to the photoelectric conversion is sampled and held in the Peter value, further A / D converted, and provided to the signal processing device as a characteristic parameter.

If the characteristic parameter is within the photographing area set before the measurement, laser light is emitted from the pulse light source 8 and the video camera 10
Next, an image of the particle specified by the characteristic parameter is captured. In the present invention, if the obtained characteristic parameter such as the scattered light intensity is within the region indicating platelet aggregation, an image is taken by considering the particles as platelet aggregation.

Analysis processing S3: A scattergram is created with the forward fluorescence intensity FL on the X-axis and the forward scattered light intensity FSC on the Y-axis, and the number of dots and the like on the scattergram in the area photographed by the detection processing. The aggregation rate is calculated by counting. The calculation of the aggregation rate will be described later.

Output processing S4: The result of the aggregation rate and the like and the scattergram calculated in the analysis processing S3 are output to a display device or a printing device. The above is the flowchart of the present invention, but the four processes (S1 to S4) are repeated for each sample, and the aggregation rate of platelets and the like are obtained for each sample. These four processes can be realized by microcomputers in the signal processing device and the image processing device.

FIG. 4 shows a flowchart of the analysis processing S3. In the image processing apparatus, the forward fluorescence intensity FL is set to X
A scattergram with the axis and the forward scattered light intensity FSC as the Y axis is developed and created on the RAM (step S5). FIG. 5 shows an example of the scattergram. Step S
At 6, the scattergram is fractionated into two areas, a red blood cell area and a platelet area. Here, as a method of fractionation,
The "two-dimensional distribution fractionation method" described in Japanese Patent Application No. 1-308964 can be used. According to this method, it is possible to draw a dividing line for dividing the erythrocyte region existing at the upper left of the scattergram and the platelet region existing at the lower part of the scattergram.

Next, in step S7, the number of dots (the number of particles) in the platelet fractionation area located below the scattergram is counted. This count is determined by a characteristic parameter provided from the signal processing device. here,
The counted number of dots is called ＄ PLT. In step S8, the number of dots ($ GATED) included in an area (referred to as an imaging control area) considered to be platelet aggregation captured by the video camera is counted. In FIG. 5, the imaging control area is an area surrounded by a thick line in the platelet area.

The photographing control area is determined as follows. First, dozens of healthy human blood without platelet aggregation,
For example, 20 samples are prepared, and after measurement using a sheath flow cell, a scattergram similar to that of FIG. 5 is drawn, and dots in the platelet region are accumulated for all 20 cases. On the accumulated scattergram, a frequency distribution of the platelet region lying on the straight line Y = X is created.

The generated frequency distribution is regarded as a lognormal distribution, and AV + 2SD (AV: average, SD: standard deviation), that is, the point of average + 2 × standard deviation, is defined as a point of the platelet aggregate on the straight line of Y = X. It is regarded as the lower limit boundary A. The region exceeding AV + 2SD contains large platelets, and it is considered that there are also aggregates formed by aggregation of single platelets.

The upper right boundary B is 0 to 25.
In the forward scattered intensity of five channels, it can be set to a point where leukocyte contamination can be avoided, for example, a point where the fluorescence intensity is 250 channels. Further, the dividing line obtained in step S6 can be set as the upper boundary of the scattergram.

The imaging control area shown in FIG. 5 captures a particle image using a sample in which platelet aggregation has been confirmed, for example, a pseudo aggregated EDTA sample, a platelet aggregation-enhancing sample, or a sample after addition of an aggregation-inducing substance. Thus, the validity can be confirmed. It should be noted that this imaging control area should be an area that includes all of the aggregates of platelets and is not an area that is composed only of aggregates.

In step S9, the captured particle images are read out one by one in order. Then, in step S10, preprocessing, for example, background correction and contour emphasis is performed, and further binarization is performed. Next, step S1
In step 1, the edge of the particle is extracted to extract the contour of the particle, and image parameters such as the particle area, equivalent circle diameter, and circularity are calculated based on the contour.

In step S12, an aggregation rate (ImgAgg%) is obtained from the obtained image parameters. For example, an aggregate of platelets can be extracted by setting an appropriate numerical value based on a criterion that the particle area is larger than a single platelet, the circularity is low, and the equivalent circle diameter is much larger than a normal platelet. Here, step S1
The processing from 0 to S12 can be performed by the same processing as described in JP-A-8-136439. However, in the case of a particle image for which it is difficult to determine the agglomerate, it is effective to use the actual visual determination of the image in combination. In step S12, the ratio of particles determined to be agglomerates in the particle images in the imaging control area and actually captured by the camera is obtained.

Next, in step S13, the aggregation rate (Agg%) in all platelets is calculated by the following equation. Agg% = (ImgAgg%) × (＄ GATED) ÷ (＄ PLT) (1) In step S14, the platelet count in whole blood (PL
T #) and the aggregate concentration (Agg #) are calculated by the following equations. PLT # (number / μL) = (＄ PLT) ÷ Analysis sample volume (μL) ÷ dilution factor (2) Agg # = (PLT #) × (Agg%) ÷ 100 (3)

In step S15, as shown in FIG. 6, the size of the agglomerate is classified into three regions according to the difference in the equivalent circle diameter for the region having a circularity of 0.85 or less. Here, particles having an equivalent circle diameter of 2 to 4 platelets correspond to small aggregates (S-Agg), and particles having an equivalent circle diameter of 4 to 10 platelets correspond to medium aggregates (M-Agg) and platelets. What corresponded to several tens or more was regarded as a large agglomerate (L-Agg), and the respective ratios when the entire agglomerate was taken as 100% were defined as an aggregation rate L-Agg.
%, M-Agg%, and S-Agg%, and multiply each by Agg # to obtain an aggregate concentration L-Agg.
#, M-Agg #, and S-Agg # are calculated. The above is the procedure for obtaining the aggregation rate and the like in the analysis of step S4.

FIG. 6 shows an embodiment of the scattergram obtained by the flow shown in FIGS. 3 and 4, the photographed image of platelets, the agglutination rate, and the like. Here, the upper part of FIG. 6 shows a scattergram and a measurement result of a negative specimen with little aggregation. In this negative sample, ImgAgg% =
0.0%, ＄ GATED = 71, ＄ PLT = 514, and the aggregation rate Agg% in the whole platelets was 0.0% from the equation (1).
%. Therefore, it can be determined that platelet aggregation hardly occurs in this negative sample. Looking at the photographed image, it can be seen that almost no aggregation is observed.

The lower part of FIG. 6 shows a scattergram and a measurement result of a positive specimen in which aggregation has appeared. In this positive sample, ImgAgg% = 44.5%, ΔG
ATED = 41, ΔPLT = 135, and the aggregation rate Agg% in all platelets is 13.5% according to equation (1). Therefore, it can be determined that platelet aggregation is high in this positive sample. It can be seen from the photographed image that there are considerable agglomerates. In addition, in this positive sample, S-Ag
g% = 65%, M-Agg% = 25%, L-Agg% =
10%. Thereby, it turns out that medium aggregates and large aggregates are considerably generated.

As described above, the scattergram is created by measuring the forward scattered light intensity and the forward fluorescence intensity, and image parameters such as circularity are obtained from the image of the particles in the set predetermined photographing control area to obtain the aggregated particles. Is performed, it is possible to detect a small aggregate of platelets whose aggregation number is about several.

The measurement shown in the flow charts of FIGS. 3 and 4 can be automated by a computer, and can be performed in a short time of about 120 seconds per sample. Therefore, the measurement of the present invention is particularly effective in the thrombus preparation state where quick measurement is desired.

The following can be considered as effective diseases in which the test according to the present invention is effective in a disease in which a thrombus preparation state occurs. 1) After surgery 2) After trauma 3) During pregnancy and puerperium 4) When taking oral contraceptives 5) Hyperlipidemia 6) Obesity 7) Extreme health care for smokers 8) Long-term bed rest 9) Malignant tumor 10) Diabetes 11) Infectious disease 12) Thrombocytosis 13) Myeloproliferative disease 14) With heart valve prosthesis 15) Ischemic heart disease 16) Cerebral thrombosis 17) Vasculitis 18) Paroxysmal nocturnal hemoglobinuria

[0048]

According to the present invention, since a scattergram is created and a particle image of a particle agglomerate in a predetermined area is taken, a quantitative measurement of the ratio of minute particle agglomerates can be performed quickly. Is possible.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of an optical system in a measuring apparatus according to the present invention.

FIG. 2 is a configuration diagram of a flow cell part in the measuring device of the present invention.

FIG. 3 is a flowchart of platelet aggregation detection according to the present invention.

FIG. 4 is a flowchart of an analysis process S3 according to the present invention.

FIG. 5 is an embodiment of a scattergram according to the present invention.

FIG. 6 is an example of a scattergram, a cohesion rate, and the like of the present invention.

[Explanation of symbols]

 Reference Signs List 5 sheath flow cell 8 pulse light source 9 projection lens 10 video camera 11 image processing device 21 continuous emission laser 22 optical fiber 23 condenser lens 24 condenser lens 25 condenser lens 26 dichroic mirror 27 photodiode 28 dichroic mirror 29 mirror 30 photomultiplier tube 31 photoelectron Intensifier

Claims (5)

[Claims] 1. A sheath flow cell for forming a flow of particles into a thin sample flow surrounded by a sheath liquid, a light irradiation unit for irradiating the sample flow with light, and detecting light obtained from the irradiated sample flow. Detecting means; calculating means for calculating a characteristic parameter of the particle from the light detected by the detecting means; determining whether or not the target particle specified by the calculated characteristic parameter is included in a preset particle aggregation region Determining means for capturing the image of the particles determined to be included in the particle aggregation region; identifying means for identifying the particle aggregate from the form of the captured particle image; and identifying means for identifying. Calculating means for calculating a particle agglomeration rate from the number of particle agglomerates and the number of target particles contained in the sample stream obtained based on the characteristic parameters. Measuring device. 2. The image processing apparatus according to claim 1, wherein the identifying unit includes an edge extracting unit that extracts a contour of the particle image, an analyzing unit that determines an image parameter that specifies a form of the particle image based on the extracted contour, 2. The platelet measurement device according to claim 1, further comprising a comparing unit that compares the obtained data with the particle aggregate data, and as a result of the comparison, a substantially identical particle is identified as a particle aggregate. 3. The particle agglutination measuring apparatus according to claim 2, wherein said analysis means obtains image parameters including an area of the particle image, an equivalent circle diameter, and a circularity. 4. The distribution means creating means for creating a distribution map corresponding to the calculated characteristic parameter, and a frequency distribution for a region including the target particle is obtained from the created distribution map. The particle aggregation measuring apparatus according to claim 1, further comprising setting means for setting a particle aggregation region based on a statistical parameter of a frequency distribution. 5. The method according to claim 1, wherein the detecting unit is formed in a first basin of the sample flow flowing through the flow cell, and after the particles detected by the detecting unit are determined to be included in the particle aggregation region by the determining unit, the particles are removed. The apparatus according to claim 1, wherein the photographing means is formed in a second basin of the sample flow so that photographing can be performed.