JPH085543A - Method for monitoring dyeing liquid for particle analysis and calibration method for particle analysis - Google Patents

Abstract

PURPOSE:To reduce influence of a change in state of dyeing liquid to particle analysis by dyeing a reference particle wherein a functional group is coupled to an artificial particle, making it flow to a flow cell, and monitoring a degree of dyeing obtained from its image. CONSTITUTION:Reference particle suspended liquid wherein a sulfonic group (functional group) is coupled to an artificial particle of styrene/divinylbenzene copolymer is dyed and injected into a flow cell 26, and the number of particles detected 37 every set time interval is counted 38. When a factor value is within a set range, 50 to 100 still images are recorded to obtain their average particle diameter. When this is within a set value, a calibration factor (a) is calculated, and further their average dyeing density is compared with a set level A. If the density is larger than the level A, it is compared with reference dyeing density of the reference particle to calculate a calibration factor (b) of the dyeing density. During routine inspection, identification parameters of size and dyeing density of the inspected particle in urine sample originated from a patient are multiplied by the factors (a), (b) respectively to provide final, parameters, with which deposit components are analyzed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a staining solution monitoring method and a calibrating method for analyzing particles suspended in a fluid.

[0002]

BACKGROUND OF THE INVENTION To analyze and classify particles in a sample of biological fluid such as blood or urine, a method of forming a smear on a glass microscope slide and flowing a particle suspension fluid through a flow cell. A flow cytometer method for analyzing is known.

As a method for solving the problem that it is difficult to classify particles due to their morphological characteristics in the flow cytometer method, a fluid sample is flown through a flow cell to obtain a static image of the particles. Is proposed.
Examples of such are described in US Pat. No. 4,338,024 and US Pat. No. 4,786,16.
There is number 5. U.S. Pat. No. 4,338,024 shows the sample flowing through a flow cell having a specially shaped passageway and a TV.
It takes still images with a camera and analyzes the particles in the sample from those images. US Patent No. 4,
No. 786,165 is an optical system and a flow that captures still images.
He teaches an optical meter for detecting the passage of particles in a cell.

On the other hand, in a flow cytometer or particle analyzer, a standard solution containing particles of immobilized erythrocytes or latex is used to check the reproducibility of the number of particles detected in the apparatus, and to use an electrical analyzer. The electrical drift and the light quantity change of the microscope are calibrated using the dynamic signal.

[0005]

In an analyzer that obtains a static image and classifies particles, biological particles in the sample are stained before the sample is introduced into the flow cell. However, since the dye in the dyeing solution is gradually oxidized or its physical properties are changed after opening, the dyeing ability is deteriorated with the passage of time.
Due to such a decrease in dyeing ability, the accuracy of analysis when performing particle analysis is reduced, and the number of particles that cannot be identified increases.

The variation in the dyeing concentration is caused not only by the temporal deterioration after the opening of the dyeing solution but also by the temperature and humidity environment and the storage condition of the day, but the operator cannot cope with the variation. Further, when the expiration of the dyeing solution or a trouble of the dyeing liquid ejection mechanism occurs, the abnormality of the device is notified only during the measurement of the sample derived from the patient.

One object of the present invention is to provide a calibration method which can reduce the influence of the change in the state of the dyeing liquid on the measurement result of the particle analysis.

Another object of the present invention is to provide a monitoring method capable of checking the particle dyeing ability in a dyeing solution.

Another object of the invention is to provide a method by which a size calibration can be carried out depending on the size of the particles to be analyzed.

Another object of the present invention is to provide a method by which it can be checked whether the flow path system through which the sample fluid flows is functioning properly.

[0011]

According to the method for monitoring a dyeing solution for particle analysis according to the present invention, a reference particle formed by binding a functional group capable of binding a dye ion to a granular base material made of an artificial substance is dyed with the dyeing solution. And flowing the dyed reference particles into a flow cell to form an image of the reference and monitoring the degree of staining obtained from the image of the reference particles.

The calibration method for particle analysis according to the present invention comprises dyeing a standard particle comprising a granular base material made of an artificial material with a functional group capable of binding to a dye ion with a dyeing solution, and dyeing the dye. Flow the reference particles to the flow cell to form the reference image, calculate the calibration coefficient based on the staining concentration information obtained from the image of the reference particles, and stain the test particles in the sample fluid. And applying the calibration factor when processing the image of the particle to be tested.

The functional groups in the reference particles are those which impart an acidic or basic character to the particulate matrix to allow the dye ions in the dye liquor to bind to the particles.
It is selected from sulfonic acid group, carboxyl group, hydroxyl group, chloro group, chloromethyl group, quaternary ammonium salt group and amino group. The base material of the reference particles is selected from polystyrene, a copolymer of styrene and divinylbenzene, a copolymer of styrene and butadiene, polyvinyltoluene and silica gel.

In a preferred embodiment of the present invention, a dye-modulating ion is added to the suspension fluid containing the reference particles so that the functional group competes with and binds to the dye ion in the dyeing solution.

In a preferred embodiment, the size of the reference particles suspended in the suspension fluid is 2 to 160 μm in diameter.
A plurality is selected from the range of m, and a calibration coefficient is calculated based on size information obtained from such an image of the reference particle, and based on the size information when processing the image of the test particle. This includes applying calibration factors.

Further, in a preferred embodiment of the present invention, a suspension fluid containing reference particles is caused to flow through the flow cell at a predetermined flow rate, and the number of reference particles flowing through the flow cell is set at predetermined time intervals. Including multiple measurements.

[0017]

The method for monitoring a dyeing solution for particle analysis according to the present invention comprises staining a reference particle, which is obtained by binding a functional group capable of binding a dye ion to a granular matrix made of artificial material, with a dyeing solution, Flowing the dyed reference particles through a flow cell to form an image of the reference and monitoring the degree of staining obtained from the image of the reference particles. Therefore, it is possible to check the particle dyeing ability of the dyeing solution.

The calibration method for particle analysis according to the present invention comprises dyeing a standard particle comprising a granular base material made of an artificial material with a functional group capable of binding to a dye ion with a dyeing solution, and dyeing the dye. Flow the reference particles to the flow cell to form the reference image, calculate the calibration coefficient based on the staining concentration information obtained from the image of the reference particles, and stain the test particles in the sample fluid. And applying the calibration factor when processing the image of the particle to be tested. This makes it possible to reduce the influence of the change in the state of the dyeing solution on the measurement result of particle analysis.

In the preferred embodiment of the present invention, the size of the reference particles suspended in the suspending fluid is 2 to 160 in diameter.
A plurality is selected from the range of μm, and a calibration coefficient is calculated based on size information obtained from the image of such a reference particle, and based on the size information when processing the image of the test particle. This includes applying calibration factors. According to this, it becomes possible to execute the calibration regarding the size according to the size of the particle.

In a preferred embodiment of the present invention, a suspension fluid containing reference particles is caused to flow through the flow cell at a predetermined flow rate, and the number of reference particles flowing through the flow cell is set a plurality of times at predetermined time intervals. Including measuring. According to this, it becomes possible to check whether or not the flow path system through which the sample fluid flows is functioning properly.

[0021]

EXAMPLE A flow path for analyzing biological particles in a sample fluid will be described with reference to FIG. First, a sample container containing a sample such as urine is set on a sample table (not shown), and "S" on the operation panel 46 (Fig. 7) is set.
Press the "TART" key. The stirring rod 1 is rotated so that the precipitated sediment component is uniform, the sampling pipette nozzle 2 is inserted into the urine sample 3, and at the same time, the syringe 4 is driven to suck a fixed amount. The dyeing liquid 14 is connected to the apparatus, the dyeing liquid pump 13 is driven, and a certain amount is discharged from the dyeing liquid discharge nozzle 12 to the dyeing tank 11 at the position a. The urine sample 3 is discharged from the pipette nozzle 2 into the staining tank 11. At that stage, the sediment component is the staining solution 14
To be dyed by.

Next, the pipette nozzle 17 for direct sampling is moved to the staining tank 11 at the position c, and at the same time, the syringe 5 is driven to suck a fixed amount of the stained urine. The nozzle 17 is moved to the flow cell 26, firmly fixed to the upper portion of the flow cell 26, and the aspirated dyed urine is injected. The sample that has passed through the flow cell 26 from the nozzle 17 drives the syringe 27 and the syringe 28, and the sheet
The sheath liquid sucked from the spray liquid 22 is caused to flow downward from both sides of the flow cell, the valves 24 and 25 are opened, and the sheath liquid is discharged to the waste liquid tank 23.

On the other hand, the sampling nozzle 2 after discharging the urine sample is moved to the 10a side of the nozzle cleaning tank 10, and the urine sample remaining in the nozzle 2 is driven by the syringe 4.
The interior of the sampling nozzle 2 is cleaned by discharging the sheath liquid supplied from the tank 22 to the nozzle cleaning tank 10 with the valve 6 open. After that, 10b of the nozzle cleaning tank 10
To the side of the sampling nozzle 2 by driving the ironing pump 21 to the side and causing the sheath liquid supplied from the tank 22 with the valve 8 opened to overflow into the cleaning tank 10 from the lower side.
Clean the outside of the.

Similarly, the nozzle 17 for direct sampling after discharging the dyed urine also has a nozzle cleaning tank 19a.
The dyed urine sample remaining in the nozzle 17 is discharged to the nozzle cleaning tank 19 together with the sheath liquid supplied from the tank 22 with the valve 5 opened by driving the syringe 5 to clean the inside of the nozzle 17. do. After that, the nozzle 17 is moved to the side 19a of the nozzle cleaning tank 19, and the syringe 5 is driven to discharge the sucked sheath liquid supplied from the tank 22 to the cleaning tank 19 to cause the overflow, and the nozzle 1
7. Wash the outside of 7.

After sucking the dyed urine, the dyeing tank 11 drives the ironing pump 21 and d with the valve 9 open.
The sheath liquid is injected from the cleaning nozzle 15 into the dyeing tank 11 at the position, and then the cleaning nozzle 16 is used to drive the ironing pump 20 with the valve 18 open to discharge the sheath liquid into the waste liquid tank 23. .

Next, a particle detecting means, a particle photographing means,
The particle analysis means will be described with reference to FIG.

The particle photographing means has a function as a microscope, causes the flash lamp 31 to emit light in a pulsed manner, and the pulsed light flux travels along the optical axis of the microscope and is focused on the sample flowing through the condenser lens 33 and inside the flow cell 26. To be done. The pulsed light flux applied to the sample flow 35 in the flow cell 26 transfers the captured image through the microscope objective lens 36 to the TV camera 39 which converts the image data signal.

The timing of flash lamp emission in the particle detecting means is controlled by the detection signal of the particle detecting system. The semiconductor laser light source 32 is always turned on, and the particle detector 37 always observes that particles in the sample pass through the detection region. If the particles pass and the particle detection signal due to the scattered light is equal to or higher than a predetermined level, the control unit 45 determines that the particles are image processing target particles, lights the flash lamp 31, and shoots an image.

The particle detecting means has a particle counter circuit 38 which counts the number of particles detected within a certain period of time, compares the number with a preset number, and transfers it to the central controller 45. Thereby, it is monitored whether the state of the sample flow is proper.

The particle analyzing means stores an image data signal transferred to the TV camera 39 into an A / D converter 40 for converting the image data signal into a digital signal, and data based on the signal from the A / D converter 40 at a predetermined address. Image memory 41, feature extraction circuit 4 for performing image processing based on signals from the image memory
2. Correction circuit 43 from image data, discrimination circuit 44 for classification, photographing condition of TV camera 39, condition for flowing sample liquid of flow cell 26, storage of processing result from discrimination circuit and display (liquid crystal display device) 47 It has a central control unit 45 for displaying and displaying on the printer 48. Further, by inputting on the operation panel 46, analysis parameter setting, data correction, mechanical system maintenance and the like are performed. In the analyzer of FIG. 7, morphological information and dyeing density information are used as identification parameters for particle analysis.

As the material of the artificial particles, polystyrene,
There are copolymers of styrene and divinylbenzene, polyvinyltoluene, silica gel, and copolymers of styrene and butadiene, and products having a particle size of 3 to 300 μm are widely marketed. In addition to spheres, multiple shapes such as ellipses and rods are used. In order to efficiently bond the functional group to the particle surface,
The surface has irregularities. It is well known as a packing material for liquid chromatography. When the surface of the latex particles is not uneven, the amount of functional groups that can be bonded is limited, the dyeing density does not increase, and dyeing takes time.

As the kind of the functional group, a sulfonic acid group (-
SO ₃ H), a hydroxyl group (-OH), a chloro group (-Cl),
Quaternary ammonium salt _{(-CH 2 N, (CH 3} ) 3 C
l), carboxyl group (-COOH), amino group (-N)
H _2), a chloromethyl group (-CH ₂ Cl) can be used.

In analyzing biological components, mainly sediment components in urine, the main sediment components include the following components.
m), red blood cells, white blood cells (6 to 20 μm), epithelial cells (2
0-100 μm), cylinder (length 100 μm-160 μm)
There is a difference in size from the above), and they are mixed in one urine sample. Therefore, the particle image analyzer takes images using two types of magnifications. Therefore, it is desirable to correct the size identification parameter in two steps.

Sternhe for dyeing solutions for urine sediment
imager-Marbin method (hereinafter abbreviated as SM method) and St
The Ernheimer modified method (hereinafter referred to as the S modified method) is in widespread use. In the SM method, the acidic dye safranin-O
(C ₂₀ H ₁₉ ClN ₄₎ and a basic dye crystal violet (C ₂₅ H ₃₀ ClN ₃₎ were mixed, in the S variant acidic dye pyronin _{B (C 42 H 52 C l8} Fe 2 N 4 O 2) And a basic dye Alcian blue (C ₃₂ H ₁₆ N ₈ Cu) are mixed and prepared. In this way, a liquid having two peak wavelengths (red for an acidic dye and blue for a basic dye) provided by an acidic dye and a basic dye is obtained, and the components can be color-coded. Other basic dyes such as Evans Blue (C _{34
H 24 N 6 Na 4 O 14} S 4), trypan blue (C ₃₄ H ₂₄ N ₆
Na ₄ O ₁₄ S ₄ ), methylene blue (C ₁₆ H ₁₈ ClN ₃ S
・ 3H ₂ O), erythrosine B (C ₂₀ H ₆ ) as an acidic dye
I ₄ Na ₂ O ₅ ). These pigments have the property of being soluble in water and dyeing sediment components when mixed with biological components of urine.

Next, preparation of an artificial particle suspension for use in calibration of particle analysis will be described.

The material of the artificial particles is a styrene / divinylbenzene copolymer particle (average particle size 8 μm) having a sulfone group (—SO ₃ H) bonded to it, which is strongly acidic and has a wet specific gravity (g / milliliter) of 1.312, It has the property of exchange capacity (meq / ml) of 2.0 or more. The other type of particles is also a particle of a styrene / divinylbenzene copolymer, the size of which is 40 μm in average particle size, and a quaternary ammonium salt group is bonded thereto. The standard particles are strongly basic and have a wet specific gravity (g / ml) of 1.312 and an exchange capacity (meq / milliliter) of 2.0 or more.

To prevent the particles from agglomerating, the particle size of 0.
1% Triton X-100 (polyoxyethylene (1
0) octyl phenyl ether) was added, and the particles became 250
The particles are suspended (suspended) so as to have a concentration of particles / microliter. This concentration is a value prepared by a particle counter. Equal amounts of these two types of particle suspensions are mixed.

A staining solution is a 2% erythrosin B aqueous solution (WM
880; 0.2 × 10 ² mol / liter) and 1% methylene blue aqueous solution (WM373.5; 2.6 × 10 ² m)
ol / l) are mixed in a ratio of 2: 1 and then the precipitate is filtered to make. The dye ion of methylene blue binds to the acidic particles of 8 μm to dye the particles blue, and erythrosin B binds to the basic particles of 25 μm to dye red purple. These two types of particles are suspended in the same fluid.

It is inexpensive to use a commercially available ion-exchange filler as the artificial particles as a material, but since many functional groups are bonded to the surface of the particles, the concentration becomes higher than that of actual biological components in urine in a short time. It tends to be dyed. Therefore, the dye-modulating ions capable of binding to the functional groups on the artificial particles are contained in the suspension fluid in advance. This dye-controlling ion competes with the dye ion of the dyeing solution and binds to the functional group of the particle, and as a result, it is possible to obtain a dyeing concentration close to the dyeing state of the biological component and to delay the dyeing time. You can

Next, referring to FIG. 1, a calibration procedure using a suspension of reference particles will be described.

The reference particle suspension is set on a sample table (not shown), the number of measurements, particle concentration (/ microliter), and average particle size (μm) are input while observing the maintenance screen, and the operation panel of the apparatus is operated. Press the start key. Mix the reference particle suspension well with the stir bar 1 and use the sampling nozzle 2
Then, a certain amount is sucked (steps 101 to 103) and discharged into the dyeing tank 11 containing the dyeing solution. Staining for the reference particles starts. After a certain period of time, the sample containing the dyed particles is sucked by the nozzle 17 and injected into the flow cell 26 (steps 104 to 105).

Thereafter, the number of particles that have passed through the particle detection region is measured at set time intervals, and if each detected number exceeds a preset range, a warning is issued and the content is displayed on the display device 47. , Inform the operator.
Details of the stability check of the flow path system will be described later (steps 106 to 108).

When the detected particles reach the image pickup area in the flow path, a still image is photographed and feature extraction of morphological information is performed. 50 to 1 due to variations in the size of artificial particles
00 static images are taken and the average value of their diameters is obtained (step 109). The average value is the average particle size entered on the maintenance screen. The correction coefficient a is calculated by comparing with the size of the image preset in the apparatus (step 110).

The shape of the artificial particle is not limited to a spherical shape, and an elliptical shape, a rod shape, or an indefinite shape can be produced. In this case, not only the diameter of the particle but also the minor axis, major axis, perimeter, area, etc. are important for morphological information. It becomes a factor. Details of the morphological information will be described later.

Next, the feature extraction of the dyeing density information of the still image is performed. Similar to the morphological information, since there are variations in the dyeing density of the particles, 50 to 100 still images are taken and the average dyeing density is obtained. If the average dyeing concentration is lower than the level set in the device (hereinafter referred to as A), the dyeing liquid is often due to the expiration of its use or a trouble with the dyeing liquid discharging mechanism. Since it is thin, the analysis accuracy does not improve even if it is corrected. So in this case,
A warning is issued to prompt the user to check the quality of the staining solution and the ejection mechanism, and the device is protected so as to prevent the start of the routine inspection (steps 111 to 112).

If the average staining density is greater than level A,
The correction coefficient b of the dyeing density is calculated by comparing with the standard dyeing density of the reference particles set inside the apparatus. (Step 1
13) Details of dyeing density correction will be described later.

Finally, the result is displayed on the maintenance screen. Average detected number concentration (/ microliter), RAN
GE, CV (%), average stain density, difference from standard stain density, and content of alarm when alarm occurs (step 1)
14). The above is the procedure of the calibration method using the reference particles.

These correction coefficients are stored, and the latest correction coefficient is adopted unless otherwise specified. It is desirable to calibrate the apparatus before the routine inspection in consideration of optical, electrical fluctuations and fluctuations in dyeing density, but the calibration operation may be performed during the routine inspection.

The routine inspection procedure will be described with reference to the flowchart of FIG. First, a patient urine sample is set on a sample table (not shown), parameters such as classification items required for analysis, sample number registration, and display unit setting are input, and the start key on the operation panel is pressed. (Steps 201 to 203) It is checked at that time whether the average staining density of the latest calibration result exceeds the level A, and if it is lower than the level A, the sampling is stopped and the stop state is set (steps 204 to 205). . If the level A is exceeded, the routine inspection is started.

A patient-derived urine sample is mixed with a stirring rod so that the sediment component and urine are uniform, and a fixed amount (800 to 1000 μl) of the sample is aspirated by the sampling nozzle 2, and a staining solution (100 to 120 μl) is added. It is discharged to the dyeing tank 11 that is being discharged, and dyeing is started. After a certain period of time, the urine dyed through the nozzle 17 flows into the flow cell 26.
(Steps 206-207).

If the detection signal of the particles that have passed through the particle detection area in the flow path is higher than a certain level, the particles are recognized as sediment components, and when the particles pass the imaging area in the flow path, the flash lamp is turned on. Lights up and shoots a still image. Feature information and dyeing density information are extracted from the still image as features (steps 208 to 209). The discrimination parameter (diameter of the particle) of the size of the test particles in the urine sample is multiplied by the correction coefficient a, and the discrimination parameter of the staining concentration is corrected by the correction coefficient b.
To obtain the final identification parameter (steps 210 to 210).
211). Each sediment component is analyzed by this final parameter, and which sediment component was present in the patient urine sample and to what extent it was displayed on the liquid crystal display device 47,
The data is printed on the printer 48 (steps 212 to 213).

In order to check the stability of the flow path system, particles are detected using the artificial particle suspension as a sample. The condition at this time is that the sample flow rate is 0.25 microliter / Se.
c, the case where the artificial particle suspension is used at a sample particle concentration of 500 particles / microliter will be described. Although there are two types of particle diameters, the number of particles is counted as one type when detecting particles. If the flow path system is stable, 125 / Se
It is expected to be about c ± 13 (± 10% variation).
However, in consideration of the particle concentration in the sample and the fluctuation of the sheath liquid flow, 125 particles / Sec ± 25 particles (± 20% fluctuation) was set as the setting range.

The actual data is shown in FIG. One of the reasons why the sample flow is not stable is that the flow cell is contaminated. Therefore, data taken for 30 days after the cleaning of the channel with the detergent was stopped is shown in FIG. White circle (○) is 1
0 days later, black triangle (▲) mark 20 days later, square (□) mark 3
After 0 days, the artificial particle suspension is flown to detect particles and
The number of detected particles was counted every second. It remains within the set range until 20 days later, and the flow path is stable. But,
After 30 days, the setting range was greatly exceeded, and the number of detections was close to 0 or 300 or more, and the fluctuation range was large and unstable. This is an example in which the flow path became unstable due to contamination of the flow cell.

The judgment criteria for the flow path system stability check are that the number of particles detected every particle detection 1 to 2 Sec is aggregated, the fluctuation range for each time is the average number ± 20% or less, and the checked number is 1 / of the average number. 3 or less was once or more, and the number of checked was 3 times or more of the average number of once or more. It is possible to check whether the flow path before the routine inspection is appropriate by issuing a warning by judgment and prompting the user to check the flow path system (flow cell, direct sampling, etc.), and cleaning of the flow path is necessary. It is determined whether or not.

FIGS. 4A and 4B are schematic views of still images taken using the reference particle suspension. When performing feature extraction of morphological information, first, 50 to 100 particle diameters of each type having different particle diameters are measured from such a static image from the image size theoretically set in the apparatus, and the average value is calculated. (8.1 μm). This average value is compared with the average particle size (8.0 μm) input on the maintenance screen, and the correction coefficient to be applied to the patient urine sample to be analyzed thereafter is calculated.
In this case, the correction coefficient a is a = 8.0 / 8.1 = 0.98.
8 and the correction coefficient b is b = 40.0 / 41.6 = 0.
It becomes 962.

As described above, the sediment components in the urine sample have different sizes, and the magnification is changed when imaging particles of 30 μm or less and particles of 30 μm or more. Particles of other sizes in the artificial particle suspension are taken in low magnification mode. The correction coefficient is calculated as in the high magnification mode. By changing the correction coefficient between the large particles and the small particles in this way, more accurate morphological information can be obtained.

The shape of the biological particles is not limited to the spherical shape,
It is diverse. When the shape of the reference particle is elliptical, rod-shaped, or indeterminate, the feature extraction is not the particle diameter, but parameters such as minor axis, major axis, perimeter, and area are extracted, and by correcting them, Analysis accuracy can be improved.

Finally, the correction of the dyeing density will be described. FIG. 5 shows the results of measuring the variation in the dyeing concentration using the reference particle suspension after opening the dyeing solution. The particles of 8 μm are dyed in blue, and the particles of 40 μm are dyed in reddish purple, and the average density per pixel of 50 to 100 particles is obtained. The staining density preset in the device is set to 100, and after opening the staining solution, 5, 10,
It was measured after 15, 20, 25 and 30 days. Depending on the components, the biological particles are dyed red with an acidic dye or blue with a basic dye. As an identification parameter for analysis, color balance is a large factor in analysis.

The dyeing solution greatly varies in dyeing ability depending on the storage state, the quality after opening, and the environment of temperature and humidity. Also, the rate of decrease in dyeing ability differs depending on the dye. The effect of temperature is large, 10
It is said that the dyeing density doubles when the temperature rises by 0 ° C.
The dye product Lot also changes subtly.

As shown in FIG. 5, the staining density gradually decreased after opening the staining solution, and the staining density also changed depending on the day. The standard concentration value preset in the device is 10
The correction coefficient b is calculated for each of the bluish and reddish so that the actual density value is 100%. For example, 10
Day blue staining density is 96.4% and correction factor b is b = 10
It becomes 0/98 = 1.04. After that, 1.04 is multiplied by the bluish stain density of the biological component to be analyzed, and the final discrimination parameter is obtained.

When the dyeing density is 70% or less of the standard density value, the analysis accuracy cannot be improved even if the correction coefficient is multiplied. The cause in this case is that the discharge amount of the stain liquid is small due to a trouble in the discharge mechanism of the stain liquid, and the expiration date of the stain liquid is often expired.
Therefore, when the density is extremely lowered in this way, it is more effective to issue a warning and prompt the user to check them.

Since the artificial particles are used as described above, not only spherical shapes but also rod shapes, elliptical shapes, and amorphous shapes can be selected according to the target component, and a plurality of sizes of 2 to 160 μm can be prepared. Moreover, it is easy to handle and store because there is no risk of infection and it does not easily deteriorate.

Further, by coloring the reference particles having a functional group bonded to the surface with a dyeing solution, the quality of the dyeing solution and the temperature environment at the time of use can be grasped. Furthermore, since the staining density reflects the staining time and the staining ability and is dyed uniformly, the staining coefficient of the static optical image is compared with the standard staining density to calculate the correction coefficient, and the staining density of the biological particle sample to be analyzed thereafter. By performing the discrimination parameter correction of, it is possible to always analyze at a constant staining density.

The reference particles in the suspension can be colored in two or more colors and can be corrected in two or more colors with one liquid due to the combination of the bound functional groups and dye ions. Since the above parameters are used, the analysis accuracy can be further improved.

[0065]

According to the present invention, the following effects can be obtained.

1. Provided is a calibration method capable of reducing the influence of a change in the state of a staining solution on the measurement result of particle analysis.

2. Provided is a monitoring method capable of checking particle dyeing ability in a dyeing solution.

3. A method is provided by which a size calibration can be performed depending on the size of the particles to be analyzed.

4. A method is provided by which it can be checked whether the flow path system through which the sample fluid flows is functioning properly.

[Brief description of drawings]

FIG. 1 is a flowchart of a calibration method in a particle analyzer according to an embodiment of the present invention.

FIG. 2 is a flow chart for performing a routine test of a sample fluid.

FIG. 3 is a graph showing an experimental result of stability check of a flow channel system.

FIG. 4 is a diagram schematically showing a still image of reference particles.

FIG. 5 is a graph showing changes in staining density after opening the staining solution.

FIG. 6 is a diagram for explaining a flow channel system of a particle analyzer for carrying out the method of the present invention.

7 is a diagram showing a schematic configuration for performing image processing in the analyzer of FIG.

[Explanation of symbols]

1 ... Stirring bar 2 ... Sampling nozzle 3 ... Sample
4 ... Syringe 5 ... Syringe 6, 7, 8, 9 ... Valve 1
0 ... Washing tank 11 ... Dyeing tank 12 ... Dyeing liquid discharge nozzle
13 ... Staining liquid pump 14 ... Staining liquid 15 ... Washing nozzle 16 ... Washing nozzle 17 ... Pipette nozzle for direct sampling 18 ... Valve 19 ... Washing tank 20
... Ironing pump 21 ... Ironing pump 22 ... Sheath liquid 23 ... Waste liquid tank 24, 25 ... Valve 26 ... Flow cell 27 ... Syringe 28 ... Syringe 31 ... Flash lamp 32 ... Semiconductor laser light source 33 ... Condenser lens 34 ... Flow cell 35 ... Sample flow 36 …
Objective lens 37 ... Particle detector 38 ... Particle counter circuit 39 ... TV camera 40 ... A / D converter 41 ... Image memory 42 ... Feature extraction circuit 43 ... Correction circuit 44
... Identification circuit 45 ... Central control unit 46 ... Operation panel
47 ... Display device 48 ... Printer

Claims (12)

[Claims] 1. A method of dyeing a reference particle, which is formed by binding a functional group capable of binding a dye ion to a granular matrix made of artificial material, with a dyeing solution, and the dyed reference particle is flowed.
A method for monitoring a staining solution for particle analysis, which comprises: flowing into a cell to form an image of the reference particle; and monitoring the degree of staining obtained from the image of the reference particle. 2. The method for monitoring a staining solution for particle analysis according to claim 1, comprising outputting an alarm when the degree of the staining does not reach a predetermined level. Liquid monitoring method. 3. Dyeing a reference particle formed by binding a functional group capable of binding to a dye ion to a granular base material made of an artificial substance with a dyeing solution, and the dyed reference particle to a flow cell. To form an image of the reference particles, to calculate a calibration coefficient based on the staining concentration information obtained from the image of the reference particles, and to dye the test particles in the sample fluid A calibration method for particle analysis, comprising applying the above calibration factor when processing an image. 4. The calibration method for particle analysis according to claim 3, wherein the functional group imparts an acidic or basic property to the granular base material. 5. The calibration method for particle analysis according to claim 4, which is selected from a sulfonic acid group, a carboxyl group, a hydroxyl group, a chloro group, a chloromethyl group, a quaternary ammonium group and an amino acid group. A calibration method for particle analysis, characterized by being present. 6. The calibration method for particle analysis according to claim 3, wherein the base material is polystyrene, a copolymer of styrene and divinylbenzene, a copolymer of styrene and butadiene, polyvinyltoluene and silica gel. A calibration method for particle analysis, characterized in that it is selected from 7. The calibration method for particle analysis according to claim 3, further comprising: a suspension fluid containing reference particles having a functional group that brings acidity and reference particles having a functional group that gives basicity. Calibration method for particle analysis, including: 8. The calibration method for particle analysis according to claim 7, wherein the suspension fluid contains a dye adjusting ion that competitively binds to the functional group and a dye ion in the dyeing solution. A method for calibrating particles, characterized by: 9. The calibration method for particle analysis according to claim 3, further comprising supplying suspended particles containing a plurality of types of reference particles having different sizes. 10. The calibration method for particle analysis according to claim 9, wherein the size of the reference particles is 2 to 1 as a diameter.
A calibration method for particle analysis, wherein a plurality of particles are selected from the range of 60 μm. 11. The particle analysis calibration method according to claim 9, wherein a calibration coefficient is calculated based on size information obtained from the image of the reference particles, and the image of the test particles is processed. A calibration method for particle analysis, characterized in that a calibration coefficient based on the size information is applied. 12. The calibration method for particle analysis according to claim 3, further comprising flowing a suspension fluid containing the reference particles into the flow cell at a predetermined flow rate, and a reference flow in the flow cell. A calibration method for particle analysis, comprising measuring the number of particles a plurality of times at predetermined time intervals.