JPH10311785A - Particle measuring equipment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To ensure an accurate measurement by describing a line for demarcating a region on a scattergram where a specified particle is present and removing a region where a non-platelet is present from a platelet region before measuring the platelet. SOLUTION: A sheath flow cell 5 forms a flow of fine sample liquid containing platelets which is illuminated with a laser light emitted from a continuous emission laser 21. Intensity signals S1-S3 of scattering light from particles and fluorescence thereof are detected by a photodiode 27 and photomultipliers 30, 31. A signal processor obtains feature parameters based on these signals S1-S3. A microcomputer produces a two-dimensional scattergram by combining the feature parameters and demarcates the erythrocyte region and the platelet region. Subsequently, a region where schistocytes are present is demarcated in the platelet region, the number of dots on the scattergram is counted along with the number of particles in each region, and then the number of high purity platelets from which the schistocytes are removed is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a particle measuring device, and more particularly to a particle measuring device for counting platelets with high accuracy.

[0002]

2. Description of the Related Art Conventionally, counting and analysis of red blood cells, white blood cells, and the like contained in blood have been actively performed using a flow cytometer. In particle measurement using a flow cytometer, a small sample stream containing the target particles is irradiated with light, and the intensity of the forward scattered light scattered by the particles and the intensity of the fluorescence are measured to count and analyze the target particles. I do.

Usually, the intensity of the obtained forward scattered light (FS
A two-dimensional scattergram is created using C) and the intensity of fluorescence (FL) as characteristic parameters, and various analysis processes are performed based on the scattergram to fractionate a region containing particles such as red blood cells and count the particles. Done. For example, since red blood cells and platelets have different sizes and shapes, they can be separated as distinct clusters on a scattergram.

[0004] Reticulated platelets are relatively large in RNA content among platelets. It is known that the number of reticulated platelets decreases in aplastic anemia, acute myeloid leukemia, and the like. Therefore, it is considered that counting and analyzing platelets in the reticulum is useful as an index of platelet formation in bone marrow of blood disease.

[0005]

As described above, platelets can be counted by using a two-dimensional scattergram prepared by using a flow cytometer. However, variations in the sensitivity of light-receiving elements for obtaining characteristic parameters are obtained. In some cases, accurate measurement was not possible. In addition, a platelet is located in the vicinity of a platelet distribution cluster (cluster) obtained by a two-dimensional scattergram by a measurement device (FIC) that captures an image of particles in a sample stream that has been trickled by a flow cytometer. It turned out to contain different particles (such as split red blood cells). Split red blood cells are fragments of red blood cells that have been torn. That is, in the conventional method, non-platelet particles such as split erythrocytes were counted as platelets, so that an accurate platelet count could not be calculated. Further, in some cases, the platelet region of the two-dimensional scattergram contains split leukocytes (including minor nuclei), which has been a cause of platelet count errors.

Therefore, the present invention has been made in view of the above circumstances, and performs accurate counting of platelets by excluding a region to which non-platelets belong from a platelet region on a two-dimensional scattergram. It is intended to provide a particle measuring device which can perform the measurement.

[0007]

According to the present invention, there is provided a sheath flow cell for forming a flow of particles into a thin sample flow surrounded by a sheath liquid, a light irradiation means for irradiating the sample flow with light,
Detecting means for detecting light obtained from the irradiated particles,
Calculating means for calculating the characteristic parameters of the particles from the detected light; distribution map generating means for generating a distribution map of the calculated characteristic parameters; and dividing the first region containing platelets in the generated distribution map A first fractionating unit that performs fractionation, a second fractionating unit that fractionates a second region that includes split erythrocytes in the first region, and a second region that is obtained by removing the second region from the first region. And a first counting means for counting the number of particles contained in the region. Here, the second fractionating means includes a distribution calculating means for calculating a frequency distribution of a distribution group in the first area including the platelets on the created distribution diagram, and a statistical parameter of the frequency distribution. And a first dividing line drawing means for setting a first dividing line for dividing the second region including the divided red blood cells.

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 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. The sheath flow cell of the present invention is a flow cell capable of forming a thin flow of a sample liquid by a hydrodynamic effect by wrapping a sample liquid containing target particles in a sheath liquid and flowing the sample liquid.
Conventionally known ones can be used. 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 types of blood cells and cells, granules or nucleic acids in cells may be reacted with a specific fluorescent reagent, and the fluorescence intensity may be measured. In particular, the present invention has a main object of accurately counting platelets contained in blood. Available fluorescent reagents include Auramine O,
Acridine orange, propidium iodide, ethidium bromide, Hoechst 33342, pyronin Y, rhodamine 123 and the like.

As the light irradiation 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, using light detection means (for example, a photodiode, a phototransistor, or a photomultiplier tube) that optically detects particles illuminated by light irradiation means and outputs intensity of light such as scattered light or fluorescence of the particles. Can be. The calculating means calculates necessary characteristic parameters such as scattered light signal intensity, fluorescent signal intensity, scattered light signal pulse width, and fluorescent signal pulse width from the detected light.

Calculation means, distribution map creation means, first, second,
Third and fourth fractionation means, first, second and third counting means,
The first and second segmentation line drawing means and the distribution calculation means are C
It can be constituted by a microcomputer consisting of PU, ROM, RAM, timer, I / O interface, etc.
The function of each means is executed by the CPU operating according to the procedure of the program stored in the RAM or the like.

The distribution chart created by the distribution chart creating means represents the distribution state of the characteristic parameters. For example, a two-dimensional scattergram in which the vertical axis represents the forward scattered light intensity and the horizontal axis represents the fluorescence intensity. It is. The intensity of each axis on the two-dimensional scattergram is represented by a relative intensity (channel) when the maximum sensitivity of the photodiode is 255 channels.

In the two-dimensional scattergram, the split erythrocytes are located in the first region containing the platelets fractionated by the first fractionating means, in the vicinity of the platelet distribution group, and on the side where the fluorescence intensity is low. In Japanese Patent Laid-Open No. 8-17
This is confirmed by an image taken with a flow cytometer disclosed in Japanese Patent No. 8826.

In addition, since it is generally known that split red blood cells cannot be expressed in a healthy person, a two-dimensional scattergram of a healthy person is prepared to fractionate a first region containing platelets,
If the distribution group of platelets is specified based on the statistical parameter of the frequency distribution, the second region where the split erythrocytes appear can be determined.

[0014] Similarly, it is known that split leukocytes do not appear in healthy individuals. When it appears, it has been confirmed by the above-described flow cytometer image photographing that it appears in a region where the fluorescence intensity of the first region including platelets is considerably large. Therefore, the area where the split leukocytes appear can also be determined by the statistical parameters of the frequency distribution of the platelet distribution population.

It is known that reticulated platelets have a larger amount of nucleic acids and granules contained therein than single platelets due to their properties. Therefore, reticulated platelets have a fluorescent intensity on a two-dimensional scattergram. Appears in an area larger than the platelet distribution population. The region including the reticulated platelets can also be determined by the statistical parameters of the frequency distribution of the platelet distribution population.

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 generation 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 generation laser light source 21 (for example, 0 mm) 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 continuously generated 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 those parameters.

The imaging of the particle image by the video camera may be performed by selecting only particles (for example, platelets) of a type designated in advance as an imaging target. 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 light source of a type that emits light only for a moment (about several tens of nanoseconds) by the light emission trigger signal Ts. 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. The video signal Vs from the video camera 10 is passed to the image processing device 11 and stored as a digital image.
Will be saved.

Further, the image processing apparatus analyzes the stored particle image and extracts the edge of the particle image, the area, the circle equivalent diameter, and the like.
Image parameters such as circularity are calculated. In the present invention, the pulse light source 8, the video camera 10, and the image processing device 11 take images of the particles and check whether the measured particles are platelets, split red blood cells, or split white blood cells. Ancillary equipment used, 2
It does not directly contribute to the creation of a dimensional scattergram or the fractionation processing of each particle region.

The parameters of characteristic parameters a to d such as the scattered light intensity and the fluorescence intensity are combined to create and display a two-dimensional scattergram.
The calculation of the number of particles is performed. The above is the outline of the configuration of the particle measuring device of the present invention.

Next, in the present invention, an embodiment will be described in which the fraction of each particle area on the scattergram and the number of platelets and the like are counted. FIG. 3 shows a flow chart of counting platelet and reticulated platelet counts of 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 the measurement is immediately mixed with 40 μL of a 2.6% auramine O solution (95.9% ethylene glycol) and 1950 μL of a 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 liquid mixed in the preprocessing is supplied to the sheath flow cell in the amount of 488 nL.
m of argon ion laser beam. 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) and the forward fluorescent light intensity (FL) are measured. You. 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.

Analysis processing S3: A two-dimensional 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 characteristic parameters measured in the detection processing on the two-dimensional scattergram Based on
The particle population is fractionated, platelets are counted, and the like. Details of this analysis processing will be described later.

Output processing S4: The result of platelet counting and the two-dimensional 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 four processes (S1 to S4)
Is repeated for each sample, and platelet counting and the like are performed for each sample. These four processes can be realized by a microcomputer in the signal processing device.

FIG. 4 shows a flowchart of the analysis processing S3. The signal processor uses the characteristic parameter of each particle obtained in the detection process S2 to calculate the forward fluorescence intensity FL.
Is created on the RAM by developing a scattergram with the X axis as the X axis and the forward scattered light intensity FSC as the Y axis (step S
5). FIG. 5 shows an example of the scattergram. The forward scattered light intensity on the vertical axis (Y axis) corresponds to the particle size. The forward fluorescence intensity on the horizontal axis (X axis) is
It corresponds to the RNA content.

In step S6, the scattergram is fractionated into two areas, a red blood cell area and a platelet area. here,
As a method for fractionation, a "two-dimensional distribution fractionation method" described in Japanese Patent Application No. 1-308964 can be used. According to this method, as shown in FIG. 5, 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.
In FIG. 5, a curve E is a dividing line.

In step S7, the distribution angle of the platelet region is calculated. Here, the distribution angle is the inclination (tan θ) of a straight line (straight line A in FIG. 5) connecting the center of gravity of the particle distribution group and the origin of the scattergram. The center of gravity of the platelet distribution population is determined from the average of the scattered light intensity (FSC) and the fluorescence intensity (FL) of each particle present in the platelet region.

In step S8, a dividing line for the split erythrocytes is determined. In the blood of healthy people without dividing red blood cells,
The distribution of platelets can be statistically estimated to fall within the mean value−standard deviation × 3 (AV−3SD) in the frequency distribution projected in the direction perpendicular to the straight line connecting the center of gravity and the origin. In addition, it has been confirmed by the photographed image that the split erythrocytes are present in a portion exceeding the AV-3SD.

Therefore, a straight line parallel to the straight line connecting the center of gravity of the platelet distribution population and the origin and corresponding to AV-3SD is determined as a dividing line for split red blood cells. Specifically, the slope tan passing through the −23 channel on the X axis indicating the fluorescence intensity
A straight line of θ is obtained, and this is set as a dividing line. In FIG.
Line B is the dividing line for split red blood cells. In FIG. 5, particles belonging to the region located on the left side of the straight line B in the platelet region are regarded as split red blood cells (Frag). The region where the split erythrocytes are present is called the Frag region.

In step S9, a dividing line for divided leukocytes is determined. It is known that split leukocytes also do not appear in healthy individuals but only appear in specimens of specific diseases. When such a sample is measured and a particle image is taken, it has been confirmed that split leukocytes appear in a region exceeding 164 channels on the X-axis indicating the fluorescence intensity.

Therefore, a straight line having a slope tan θ passing through the 164 channels on the X axis is obtained, and this is defined as a dividing line of the split leukocyte. In FIG. 5, a straight line C is the dividing line, and particles belonging to a region located on the right side of the straight line C in the platelet region are regarded as split leukocytes (Other). The region where the split leukocytes are present is called the Other region.

The region containing high-purity platelets is specified by the platelet region obtained in steps S6, S8 and S9 and the dividing lines B and C.
That is, of the particles contained in the platelet region, Fra
It can be determined that high-purity platelets are obtained by removing particles in the g region and particles in the Other region.

Next, in step S10, a dividing line for reticulated platelets is determined. It is known that reticulated platelets have a fluorescence intensity that is higher than that of normal single platelets,
Here, the average value of the frequency distribution described above + standard deviation × 2
A straight line at a position corresponding to (AV + 2SD) is defined as a dividing line for reticulated platelets.

Specifically, a straight line having an inclination tanθ passing through 20 channels on the X-axis of the scattergram is defined as a dividing line,
Platelets located on the right side of this straight line are determined to be reticulated platelets. In FIG. 5, a straight line D is the dividing line, and particles belonging to a region surrounded by the straight lines D and C in the platelet region are regarded as reticulated platelets (RP). The region where the reticulated platelets are located is called the RP region. As described above, particles on the scattergram are divided into the Frag region, the RP region, and the O region.
It is fractionated into a ther region and a region of a platelet distribution population.

FIG. 6 shows an embodiment of a scattergram of a sample in which split red blood cells have appeared. FIG. 6A shows a scattergram in which the red blood cell region and the platelet region are fractionated, and FIG. 6B shows a scattergram after removing the Frag region from FIG. 6A. . A region surrounded by straight lines B and C in the platelet region is a platelet region of high purity.

Next, in step S11, particles in each area are counted. Particle counting is performed by the CPU counting the number of dots on the scattergram developed on the RAM for each area. Where Fr
The count values of the number of dots in the ag area, the RP area, and the Other area are calculated as $ Frag, $ RP and $ Other, respectively.
Call.

In step S12, the ratio of the number of particles in each region to the total number of particles in the platelet region is determined from the above count value and the total number of dots in the platelet region (領域 PLT) by the following equation. . Frag% = ＄ Frag / ＄ PLT × 100 (1) RP% = ＄ RP / ＄ PLT × 100 (2) Other% = ＄ Other / ＄ PLT × 100 (3) where Frag% Is the ratio of split erythrocytes to the total platelet count, RP% is the ratio of reticulated platelets to the total platelet count, O
ther% represents the ratio of split leukocytes to the total platelet count.

The platelet concentration PLT # in whole blood
(Number / μL) is calculated by the following formula using the analyzed sample volume and dilution ratio. PLT # = {PLT} analysis sample volume (μL) / dilution factor (4) Furthermore, the split erythrocyte concentration in whole blood (Frag #),
Retinal platelet concentration (RP #) and split leukocyte concentration (Othe
r #) is calculated by the following equation. Frag # = PLT # × Frag% ÷ 100 (5) RP # = PLT # × RP% ÷ 100 (6) Other # = PLT # × Other% ÷ 100 (7) In addition, split erythrocytes and The number of high-purity platelets excluding split leukocytes (P-PLT #) is determined by the following equation. P-PLT # = PLT # -Frag # -Other # (8)

Each count value obtained by performing the analysis processing of the present invention for the sample shown in FIG. 6 is shown below. ＄ PLT = 1059 (dots) # PLT = 195 × 10 ³ (pcs / μL) RP% = 0.27 (%) Frag% = 0.5 (%) Other% = 0.0 (%) RP # = 0 .53 × 10 ³ (pieces / μL) Frag # = 0.98 × 10 ³ (pieces / μL) Other # = 0.0 (pieces / μL) P-PLT # = 194 × 10 ³ (pieces / μL) By the analysis as described above, the high-purity platelet count (P-PL
T #) can be counted, and the number of reticulocytes (R
P #) can also be calculated.

[0044]

According to the present invention, since non-platelet particles such as split erythrocytes are removed from the platelet region by drawing a demarcation line on the scattergram to separate the region where the predetermined particles exist. Accurate counting of platelets 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 fractionation / counting of 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 of a sample in which split erythrocytes of the present invention appear.

[Explanation of symbols]

 Reference Signs List 5 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 photomultiplier Doubler

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 a detection unit for detecting light obtained from the irradiated particles. Calculating means for calculating characteristic parameters of particles from the detected light; distribution map generating means for generating a distribution map of the calculated characteristic parameters; and a first area including platelets in the generated distribution map. First fractionating means for fractionating, and a second fraction containing split erythrocytes in the first region.
And a first counting unit that counts the number of particles contained in a region obtained by removing the second region from the first region. Particle measurement device. 2. A distribution calculating means for obtaining a frequency distribution of a distribution group in a first area including platelets on a created distribution map, wherein the second fractionating means includes a statistical parameter of the frequency distribution. 2. A particle measuring apparatus according to claim 1, further comprising: a first segmentation line drawing unit for setting a first segmentation line for segmenting a second region including the divided red blood cells based on the first segmentation line. . 3. A third fractionating means for fractionating a third region containing divided leukocytes in the first region,
2. The particle measuring apparatus according to claim 1, further comprising a second counting unit configured to remove the number of particles in the third region from the number of particles counted by the counting unit. 4. The method according to claim 4, wherein the fourth area is divided into a fourth area including reticulated platelets in the first area, and the fourth area is determined based on the number of particles counted by the second counting section. 4. The particle measuring apparatus according to claim 3, further comprising third counting means for counting only the number of particles included in the region. 5. A second dividing line for setting a second dividing line for dividing a fourth region containing reticulated platelets based on the statistical parameter of the frequency distribution. The particle measuring device according to claim 4, further comprising a drawing unit.