JPH06194364A - White blood cell classification reagent in flow cytometry - Google Patents

Abstract

PURPOSE:To classify and count white blood cells through a simple procedure and constitution by composing a reagent of a dye for selectively fluorochroming both acidocyte and basocyte, a dye for fluorochroming the nucleus of white blood cell, a buffer for holding pH, and an osmotic pressure compensation agent for adjusting osmotic pressure. CONSTITUTION:A reagent is composed of a dye for fluorochroming both acidocyte and basocyte selectively, a dye for fluorochroming the nucleus of white blood cell, a buffer for holding pH at 6.0-11.0, and an osmotic pressure compensation agent for conditioning a dye solution to an osmotic pressure for keeping the shape of white blood cell. In the flow cytometer light emitted from a laser 12 is condensed by a cylindrical lens 16 and cast on a sample flowing through a flow cell 14. When a stained white blood cell in the sample is irradiated with the laser beam, scattering light and fluorescence are emitted from the white blood cell. Scattering light and fluorescence emitted sideways are collected by a condenser leans 18 and sent through dichroic mirrors 22, 30 to photomultipliers 36, 42 where they are measured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent used for classification of blood cells in the field of clinical examination, and more specifically, it optically measures blood cells subjected to fluorescent staining using a flow cytometer. , Classify white blood cells 2
It relates to reagents used in one of the methods.

[0002]

2. Description of the Related Art Leukocytes in the peripheral blood of healthy people include lymphocytes, monocytes, neutrophils, eosinophils and basophils. These have different functions, and by counting leukocytes in blood by type, they can contribute to diagnosis of diseases. For example, an increase in neutrophils is seen in inflammation, myocardial infarction, leukemia, etc., and a decrease in neutrophils is seen in viral diseases, aplastic anemia, agranulocytosis, etc. Increased eosinophils are found in parasitic diseases, Hodgkin's disease, and allergic diseases. The increase of monocytes is seen in the recovery stage of infection, monocytic leukemia, etc.

[0003] The most conventionally practiced method for classifying and counting white blood cells is called blood image examination (comparative method, manual method).

In this method, blood is smeared on a slide glass, blood cells are fixed, further stained, and then observed with a microscope. Morphological characteristics of each leukocyte (size of leukocyte, morphology of nucleus, Based on the cytoplasm morphology, the presence or absence of granules, etc.) and the degree of staining, it is determined which blood cell the examiner is, and the classification is
It is to count. At this time, 10
0 to 200 white blood cells were counted, and the percentage (%) of each blood cell in the total number of white blood cells was used as the measurement value.

This method requires complicated sample preparation operations such as smearing, fixing and staining of blood before observation with a microscope, and classification-counting using a microscope shows a slight difference in blood cells. It is a great burden on the measurer due to the fact that it must be distinguished. Further, since the number of white blood cells to be counted is small and the blood cells on the smear sample may have an uneven distribution, it is difficult for even a skilled measurer to give a reproducible measurement value.

For this reason, there is a strong demand for a method capable of automatically classifying and counting white blood cells.
There are roughly two types of methods implemented.

One of the methods is to classify white blood cells by capturing a blood cell image with a video camera or the like and performing image processing by a computer. This method is similar in principle to the conventional disparity method, but many blood cells cannot be classified by computer processing, and it is not a complete alternative to disparity method. There is also a problem that the device becomes complicated and large, and the price becomes high.

Another method for automatically classifying and counting white blood cells is a method using a flow system. This method uses a sample in which blood cells are suspended in a diluent, and the sample is flowed so that each blood cell passes through a thin detector one by one, and white blood cells are detected by analyzing the signal generated by the detector at this time. It is to classify. The method utilizing this flow system is further subdivided into two methods.

The first method is to destroy red blood cells with a lysing agent,
An electrolytic solution in which only white blood cells are suspended is flown into the fine pores, impedance changes in the fine pores when blood cells pass through the fine pores are detected, and the white blood cells are classified according to the magnitude of the detection signal.

In the second method, the light source and the cells in the sample are
A flow cytometer equipped with a flow cell configured to flow through a narrow flow path one by one, a photometric unit for detecting light emitted from cells, and an analyzer for analyzing a detection signal is used. This method involves staining blood cells, irradiating the stained blood cells with light, detecting the fluorescence emitted from the blood cells and, in some cases, scattered light together, and classifying white blood cells by the intensity of the detection signal. is there.

Examples of the method belonging to the second method include, for example, Japanese Examined Patent Publication No. 59-853 and LA Kamensky, "Brad.
"Cells", Volume 6, 121
~ 140, 1980. This method involves adding 10 times the amount of acridine orange staining solution to blood, incubating for 1 minute, and then measuring the green fluorescence and red fluorescence emitted from blood cells when irradiated with a light source such as an argon ion laser. , White blood cells are classified and counted.

Other examples belonging to the second method include Japanese Patent Laid-Open No. 50-20820 and HM Shapiro et al., "The Journal of Histochemistry and Site". Chemistry (The Journal of Histochemi
second story and Cytochemistry)
Vol. 4, No. 1, 396-411, 1976; and also Vol. 25, No. 8, page 976-989, 1977. This method uses 4 times the amount of stain 1 for blood.
After incubating for 3 minutes, add 2
0% formaldehyde is added, fixed for 5 minutes, diluted 15 to 20 times with Staining Solution II for dilution, and measured with a flow cytometer. The flow cytometer used for this measurement is equipped with a mercury arc lamp that divides light into three or three lasers as a light source, and each excites three types of fluorescent dyes contained in the dyeing solution, and the three types of fluorescence and forward It is an apparatus that measures 6 parameters of scattered light, side scattered light, and absorption and classifies and counts white blood cells by four-step two-dimensional distribution analysis.

Further, Japanese Patent Application No. 61-213715 filed on Sep. 10, 1986 discloses a one-step dyeing process in which blood is added to a dyeing solution consisting of a buffer solution, an inorganic salt and a fluorescent dye to dye. However, unlysed red blood cells may affect the measurement data, resulting in unclear measurement.

[0014]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention In the first method of classifying and counting white blood cells using a flow system, erythrocytes must be destroyed. In some cases, the measurement cannot be performed, and at this time, there is a problem that the accuracy of the measured value is impaired.

Among the second methods using the flow system, the method described in Japanese Patent Publication No. 59-853 is
Before the dye's absorption by the cells reaches equilibrium, that is,
It is characterized in that it is measured at the time when the difference in the fluorescence intensity of each white blood cell becomes maximum during the staining. However, for samples with extremely high or low white blood cell count, the staining time at which the staining intensity is at an appropriate level will be different from that of normal samples, and an appropriate staining time must be selected for each sample. I won't. In addition, since this method attempts to classify white blood cells only by the difference in fluorescence intensity, there is a problem that the separation of each blood cell such as the separation of lymphocytes and monocytes is not necessarily good.

Another example of the second method utilizing the flow system, that is, the method described in Japanese Patent Laid-Open No. 50-20820, has many operating procedures, takes a long dyeing time, and is complicated. Reagents must be used. Moreover, in addition to the need for three types of light sources, six parameters must be measured, which makes the device very complicated and expensive. Furthermore, since many parameters are measured in this way, the analysis becomes complicated, and there is a problem that a large-capacity analysis device is required.

Further, the above-mentioned Japanese Patent Application No. 61-213715.
In the method of classifying white blood cells by flow cytometry including the invention of No. 6, there is a problem that when the neutrophils become dead cells because the blood is left for a long time, it may not be detected by the flow cytometer. It was For this reason, fresh blood must be used as the measurement blood.

The present invention has been made to solve the above-mentioned conventional problems, and provides a reagent for accurately classifying and counting leukocytes with a simple procedure and configuration.

[0019]

[Means and Actions for Solving Problems] There are two methods for classifying leukocytes using the reagent of the present invention, each of which is hereinafter referred to as a one-step method or a two-step method.

The one-step method comprises the following steps (a) to (c).

(A) A dye which selectively fluorescently stains both eosinophils and basophils, a dye which fluorescently stains leukocyte nuclei, and a buffering agent for keeping the pH at 6.0 to 11.0. And an osmotic pressure compensating agent to adjust the staining solution to an osmotic pressure that maintains the morphology of white blood cells, add anticoagulated blood to the staining solution, and stain until equilibrium is reached. A step of adjusting the sample, (b) flowing the measurement sample into a flow cytometer, distinguishing leukocytes from other blood cells by fluorescence intensity, and determining a side (90 °) scattered light signal of leukocytes and a fluorescence signal. A step of measuring, (c) a step of discriminating and counting the type of each white blood cell based on the side scattered light signal and the fluorescence signal emitted from the white blood cell, and calculating the ratio of each white blood cell.

The two-step method comprises the following steps (a) to (d).

(A) Hypotonicity consisting of a dye that selectively fluorescently stains both eosinophils and basophils, a dye that fluorescently stains the nuclei of white blood cells, and a buffering agent for keeping the pH in an acidic range. A step of adding anticoagulated blood to the first liquid to hemolyze erythrocytes, and (b) a buffer for neutralizing the acid in the buffer of the first liquid to keep the solution pH at the staining pH. A second solution comprising an agent and an osmotic pressure compensating agent for adjusting the solution to an osmotic pressure that maintains the morphology of white blood cells, is added to the blood sample treated with the first solution obtained in (a) above. And (c) flowing the stained sample in a flow cytometer to distinguish leukocytes from other blood cells and ghosts by fluorescence intensity, and scattering the leukocyte fluorescence signal and side (90 °) scattering. Measuring the optical signal, (d) the fluorescent signal emitted from leukocytes and the side The scattered light signal, determines the type of each white blood cell counting, and calculates the ratio of each leukocyte steps.

The reagent used in the present invention for classifying white blood cells by flow cytometry has the following composition.

(1) A dye that selectively fluorescently stains both eosinophils and basophils, for example, Astrazone Yellow 3G (2) One of the following dyes that fluorescently stain the nuclei of white blood cells.

Acridine Red Rhodamine S Rhodamine 6G Rhodamine B Rhodamine 19 Perchlorate Rhodamine 123 Eosin Y Cyanocin Cresyl First Violet Darrow Red Acronol Phloxine FFS 1,1'-Dimethylthiocarbocyanine 1,1'-Diethylthiocarbocyanine 1, 1'-Diethyl-9-methylthiocarbocyanine bromide 2- [γ- (1'-ethyl-4 ', 5'-benzothiazolilidene) propenyl] -1-ethyl-4,5-benzoxazolidium iodide Astrazone Red 6B Basic Violet 16 2- (p-Dimethylaminostyryl) -1-ethyl-
4,5-Benzothiazolium iodide 2,4-bis (p-dimethylaminostyryl) -1-ethyl-pyridinium iodide 2,6-bis (p-dimethylaminostyryl) -1-ethyl-pyridinium iodide TA-2 (Japan Photosensitive Dye Research Institute: Okayama City) Astrazone Orange R (especially Astrazone Orange R)
Is preferred. ) Now, of the fluorescence signals and side scattered light signals emitted from white blood cells, the fluorescence signals reflect the cytochemical characteristics, and due to the interaction between the dye and each white blood cell, a different intensity from each white blood cell The signal is obtained.

On the other hand, the side scattered light signal reflects intracellular information. That is, the larger the cell nucleus in the cell and the more the granules, the stronger the reflection of light in the cell and the more the side scattered light intensity increases. Therefore, lymphocytes have no or few granules inside them,
The scattered light intensity is the smallest, and since neutrophils have many granules inside and have large nuclei, the scattered light intensity is large. The scattered light intensity of eosinophils is almost comparable to that of neutrophils. The intensity of light scattered by monocytes and basophils is between lymphocytes and neutrophils. For this reason, the relative intensity of the side scattered light of each white blood cell is as shown in FIG.

Therefore, by combining the fluorescence signal and the side scattered light signal, it becomes possible to classify the white blood cells into five, as will be described in detail later.

As described above, the method of the present invention does not require complicated pretreatment and the like, and only one step or two steps of simple staining are used to classify and classify only leukocytes in blood by a flow cytometer. It is to count.

A specific example of the optical system of the flow cytometer used in the present invention will be described with reference to the drawing shown in FIG. FIG. 1 shows a case where side scattered light, red fluorescence and green fluorescence are measured. The light source used for the optical system 10 of this flow cytometer is an argon ion laser 12 having a wavelength of 488 nm and an output of 10 mW. The light emitted from the laser 12 is focused by the cylindrical lens 16 and illuminates the measurement sample flowing in the flow cell 14.

When the stained white blood cells in the measurement sample are irradiated with laser light, scattered light and fluorescence are emitted from the white blood cells.

Of these, the scattered light and fluorescence emitted laterally are collected by the condenser lens 18, pass through the aperture 20, and then reach the dichroic mirror 22.

The dichroic mirror 22 receives the side scattered light 2
4 is reflected and the fluorescence 26 is transmitted. The side scattered light 24 reflected by the dichroic mirror 22 is measured by the photomultiplier tube 28. Dichroic mirror 2
Of the fluorescence 26 that has passed through 2, red fluorescence 32 is reflected by the dichroic mirror 30, and only green fluorescence 38 is transmitted. The reflected red fluorescence 32 passes through the color filter 34, and then is measured by the photomultiplier tube 36. The transmitted green fluorescence 38 is a color filter 40.
After passing through, the measurement is carried out by the photomultiplier tube 42.

By the way, since red blood cells in the sample for measurement emit only very weak fluorescence, as long as the fluorescence intensity is measured, even if the red blood cells pass through the detection unit at the same time as the white blood cells (simultaneous passage), the white blood cells are measured. Does not interfere with. However, when measuring scattered light, red blood cells emit scattered light of the same level as white blood cells, and thus interfere with counting of white blood cells. At this time, fluorescence and scattered light can be measured at the same time, and only white blood cells that emit fluorescence with a certain level of intensity or more can be taken as white blood cells. Since the light is superimposed, the correct scattered light intensity of white blood cells cannot be measured. In the optical system 10 of the flow cytometer shown in FIG. 1, the dilution ratio is set to, for example, 20 times to reduce the probability of simultaneous passage of red blood cells and white blood cells, and to make the degree of interference by red blood cells practically negligible. The held measurement sample is caused to flow in the flow cell 14.

However, the remaining white blood cells or lymphocytes after excluding eosinophils and basophils by fluorescence intensity,
When the intensity distribution of the side scattered light signals of monocytes and neutrophils is drawn, these three populations are not completely separated, as shown in FIG.

If the dilution ratio of the sample for measurement is further increased and the probability of simultaneous passage of red blood cells and white blood cells is suppressed to such an extent that the interference by red blood cells can be completely ignored, lymphocytes, monocytes, and neutrophils can be used. The frequency distribution of the side scattered light signal intensity is 4th
As shown in the figure, these three can be completely separated. However, in order to ensure the precision of the measured value, it is necessary to measure about 10,000 white blood cells, so if the dilution ratio is increased to dilute the sample, the measurement time will be too long and not practical.

Although the above problem of interference by red blood cells can be solved by performing a red blood cell removal operation such as red blood cell hemolysis treatment on the measurement sample, in the prior art, there are red blood cell removal methods such as the red blood cell hemolysis method that meet the staining conditions. I couldn't do it because I didn't. There is no prior art example in which hemolysis is performed by classifying white blood cells into five according to fluorescent staining. Further, there has been no method for hemolyzing only red blood cells within 1 minute and not impairing the side scattered light (morphological information) of white blood cells.

Generally, the following method is known for preparing a white blood cell measurement sample from which red blood cells have been removed.

I) Red blood cell hemolysis method a) Surfactant treatment b) Ammonium salt (eg NH ₄ Cl) treatment c) Hypotonic treatment (physiological pH) ii) Separation method d) Centrifugation e) Sedimentation f) Others The above (a) to (e) will be described below.

A) Surfactant treatment inhibits staining,
Simultaneously with erythrocyte hemolysis, morphological changes such as nucleation, swelling, and contraction of leukocytes occur, which makes it difficult to fractionate leukocytes by scattered light signals, and changes in leukocyte morphology over time.

B) Ammonium salt treatment: There is a problem that it inhibits staining, has a low ability to hemolyse erythrocytes, for example, it is difficult to prepare a concentrated sample obtained by diluting whole blood by 20 times, and it takes time (3 to 5 minutes) for erythrocyte hemolysis. is there.

C) Hypotonic treatment Generally, in a hypotonic solution, the resistance of white blood cells is higher than that of red blood cells, so that only red blood cells are hemolyzed and only white blood cells are left. Under the condition of complete hemolysis, some white blood cells are also destroyed.

D) and e) Centrifugal Separation and Sedimentation Separation: The operation is complicated and time consuming.

There are problems such as loss of white blood cells and fluctuation of fractionation ratio.

In the two-step method of the present invention, red blood cells in the sample are destroyed by acid hypotonic treatment in order to reduce the above-mentioned disturbance of the side scattered light intensity distribution due to simultaneous passage of red blood cells and white blood cells.

As described above, when the hypotonic treatment is carried out in the physiological pH range, some red blood cells are destroyed at the same time as the red blood cells are destroyed.

When the hypotonic treatment is carried out in an acidic pH range, particularly pH 2.0 to 5.0, leukocytes are completely retained and only erythrocytes are destroyed. In this case, morphological changes such as nucleation, swelling and contraction of leukocytes do not occur.

Erythrocyte selection The mechanism of action of hemolysis is unknown, but probably due to hypotonic treatment. It is considered that erythrocyte weakening and leukocyte acid fixation due to acidic pH proceed with the progress of erythrocyte hemolysis, and only leukocytes having resistance as compared with erythrocytes remain.

Red blood cells are ghosted and partially fragmented by the acidic hypotonic treatment. As a result, the red blood cell side scattered light signal intensity is 1 of the lymphocyte side scattered light signal intensity.
/ 2 to 1/3 or less, and the simultaneous passage of red blood cells-white blood cells becomes negligible.

However, in the acidic hypotonic treatment, since all red blood cells are not fragmented, it is difficult to completely discriminate red blood cells from white blood cells only by the scattered light signal intensity.

Therefore, the distinction between red blood cells and white blood cells is
As described above, it is desirable to use the fluorescence signal intensity.

Next, the function of the dye will be described.

The chemical structural formula of Astrazone Yellow 3G is as follows.

[0054]

[Chemical 1] The excitation-fluorescence characteristics of Astrazone Yellow 3G are shown in FIG. Excitation maximum is 430-455 nm, fluorescence maximum is 52
It is 5 nm.

Astrazone Yellow 3G binds to eosinophil and basophil internal substances such as heparin, histamine, histone and protamine and strongly stains granules.

A two-dimensional distribution map of fluorescence intensity and side scattered light intensity was drawn, showing that Astrazone Yellow 3G had a low concentration (5
At 0 to 100 ppm), only basophils are in the measurable range as shown in FIG. The fluorescence intensity of basophils is 100-
It becomes a constant value at 200 ppm, and does not increase beyond that.

Astrazone Yellow 3G density 100-2
At 00 ppm, eosinophils can be measured, and the fluorescence intensity increases as the dye concentration increases, as shown in FIG.

As shown in FIG. 7, with the dye of Astrazone Yellow 3G only, lymphocytes without granules could not be stained, lymphocytes, monocytes and neutrophils could not be stained, and total leukocytes were measured. Therefore, it is necessary to add a dye that stains the nuclei of all white blood cells without impairing the staining performance of Astrazone Yellow 3G. The above-mentioned dyes that classify white blood cells into lymphocytes, monocytes, and granulocytes all meet this purpose, but particularly when the argon ion laser is used as the light source 12 of the flow cytometer, the excitation maximum is obtained. Is 48
Dyes around 8 nm are preferred. The dye thus selected is Astrazone Orange R.

The chemical structural formula of Astrazone Orange R is as follows.

[0060]

[Chemical 2] The excitation and fluorescence characteristics of Astrazone Orange R are shown in FIG. The excitation maximum is 490 nm and the fluorescence maximum is 520 nm.

FIG. 9 shows a two-dimensional distribution chart by fluorescence and side scattered light when white blood cells are classified using a dye in which Astrazone Yellow 3G and Astrazone Orange R are combined. In FIG. 9, FL is the relative fluorescence intensity,
Side Sc indicates relative intensity of side scattered light, 1 indicates lymphocytes, 2 indicates monocytes, 3 indicates neutrophils, 4 indicates eosinophils, and 5 indicates basophils. 6 represents a noise component (hereinafter the same).

As shown in FIG. 9, white blood cells can be classified into 5 types according to the fluorescence intensity and the side scattered light intensity. As described above, when the dye in which Astrazone Yellow 3G and Astrazone Orange R are combined is used, only one channel needs to be measured as the fluorescence signal, so that the dichroic light in the optical system of the first flow cytometer can be used. Mirror 30,
The color filter 34 and the photomultiplier tube 36 are unnecessary, and the structure is simpler than that of the device used in the specific example of Japanese Patent Application No. 61-2313715.

Next, the above-mentioned one-step method and two-step method regarding the composition, pH and osmotic pressure of the solution will be described in detail.

One-Step Method (a) Dye Concentration Astrazone Yellow 3G Concentration 5 of 50-400 ppm
Level and Astrazone Orange R concentration 100-400pp
FIG. 10 and FIG. 11 show the relative fluorescence intensities of eosinophils and basophils at each dye concentration of 20 sets in combination with 4 levels of m. The results for basophils and eosinophils are shown in solid and dotted lines in FIG. 10, and the results for lymphocytes / neutrophils and monocytes are shown in solid and dotted lines in FIG. 11, respectively. . From FIG. 10, the intensity of basophils increased more than that of other leukocytes at the Astrazone Yellow 3G concentration of 100 ppm or more, and the intensity of eosinophils increased at 200 ppm or more. It can be seen that the intensity of No. 2 hardly increases as the concentration of Astrazone Yellow 3G increases. Therefore, in order to separate basophils and eosinophils from other leukocytes by fluorescence intensity, Astrazone Yellow 3G concentration should be 1
It is understood that the amount should be 50 to 200 ppm or more.

FIG. 12 shows the dependency of the fluorescence intensity of lymphocytes / neutrophils and the intensity of noise at the Astrazone Yellow 3G concentration of 300 ppm on the Astrazone Orange R concentration. Astrazone Orange R concentration 100ppm
As described above, the fluorescence intensity of lymphocytes / neutrophils and noise can be separated, and the separation becomes good at 200 ppm or more. Above 300 ppm, the separation between lymphocytes / neutrophils and noise becomes almost constant, and the fluorescence intensity of basophils and eosinophils slightly decreases with increasing Astrazone Orange R concentration. The orange R concentration is preferably about 300 ppm.

(B) pH The change in the fluorescence intensity ratio of basophils to neutrophils and eosinophils to neutrophils when the pH of the staining solution was changed between 7.5 and 9.5. They are shown in FIGS. 13 and 14, respectively. From the figure, it can be seen that the highest fluorescence intensity ratio of basophils and eosinophils to neutrophils is at pH 8.5 to 9.0.

(C) Type of Buffering Agent As the buffering agent, glycylglycine, taurine, boric acid, trycin, etc. can be used, and among them, trycin 10-10 mM is preferably used.

(D) Osmotic pressure compensating agent The fluorescence intensity does not change at a sodium chloride concentration of 75 mM to 225 mM. It may be used near isotonicity (150 mM).

Regarding the two-step method dye concentration, the final concentration of the two-step method may be set within the concentration range of the one-step method. Other conditions will be described.

(A) First Liquid pH The first liquid pH is preferably 5.0 or less for hemolyzing red blood cells, but 3.5 or more is required for preventing platelet aggregation. A pH of 4.5 is particularly suitable.

(B) First Solution Buffering Agent As the first solution buffering agent, any buffering agent having a pKa of about 4.5 such as citric acid, maleic acid or diglycolic acid can be used. For example, when diglycolic acid is used, when the concentration of diglycolic acid is 5 mM or less, the fluorescence intensity of basophils is slightly reduced, and when it is 50 mM or more, poor hemolysis of red blood cells is observed. The optimal diglycolic acid concentration is about 10 mM.

(C) Second liquid osmotic pressure The final osmotic pressure is 167 to 387 m by changing the amount of the osmotic pressure compensating agent (for example, sodium chloride) added to the second liquid.
There is no change in the fractionation pattern even if it is changed between Osm / kg. The final osmotic pressure of the second liquid is almost isotonic (280 mO
sm / kg).

(D) Second-liquid buffering agent As the second-liquid buffering agent, a buffering agent having a pKa of 8.5 to 9.0, such as boric acid, Tris, or tricine, can be used.
For example, when using Tricine, the concentration of Tricine is 50
Below mM, the fluorescence intensity of basophils and eosinophils decreases. The preferred tricine concentration is 300 mM.

(Example) An example in which the present invention is carried out under the most preferable conditions within the above-mentioned composition range is shown below.

Example 1 One-Step Method Reagent Composition Astrazone Yellow 3G (basophil and eosinophil specific dye) 350 ppm Astrazone Orange R (dye for fluorescent staining of leukocyte nuclei) 300 ppm Tricine-sodium hydroxide (buffer) Agent) 30 mM sodium chloride (osmotic pressure adjusting agent) 115 mM pH 8.7, osmotic pressure 280 mOsm / kg Staining method 1 part by volume EDTA2K 20 parts by volume of staining solution is added to anticoagulated blood, and after stirring, at 25 ° C. for about 1 minute Incubate.

Selection of filter and dichroic mirror Dichroic mirror 22 510 nm (transmits light not reflected but reflected wavelengths of 510 nm or less) Color filter 40 520 nm (transmits wavelengths of 520 nm or more) Analysis results Under the above conditions Then, using a flow cytometer, a two-dimensional distribution map based on the fluorescence intensity and the side scattered light intensity is drawn.
It becomes like the figure. As shown in the figure, white blood cells are classified into 5 categories, but there is some overlap in distribution between lymphocytes and neutrophils. This is because the side scattered light is disturbed by the effect of simultaneous passage of red blood cells and white blood cells.

Example 2: Two-step method Reagent composition First solution Astrazone Yellow 3G (basophil and eosinophil specific dye) 385 ppm Astrazone Orange R (dye for fluorescent staining of leukocyte nuclei) 330 ppm Diglycolic acid- Sodium hydroxide (buffer) 10 mM pH 4.5, osmotic pressure 50 mOsm / kg second solution Tricine-sodium hydroxide (buffer) 300 mM sodium chloride (osmotic pressure adjusting agent) 750 mM pH 9.8 to 9.9, osmosis Pressure 2200 mOsm / kg Color development method 1 part by volume of EDTA2K 18 parts by volume of the first solution was added to anticoagulated blood, and after stirring and incubation at 25 ° C. for 20 seconds, 2 parts by volume of the second solution was added, and after stirring, 25 ° C. At 40
Incubate for seconds. The final dyeing condition is pH 8.6-
8.7, osmotic pressure 286 mOsm / kg (isotonic).

Selection of filter and dichroic mirror Same as in the first embodiment.

Analysis Results Under the above-mentioned conditions, a two-dimensional distribution map based on the fluorescence intensity and the side scattered light intensity measured by a flow cytometer was drawn.
It becomes like the figure. As shown in the figure, the white blood cells were classified into 5 categories, and the slight overlap of distributions observed between lymphocytes and neutrophils in Example 1 did not appear. This is because erythrocytes were hemolyzed, and the effect of simultaneous passage of erythrocytes and leukocytes was completely eliminated.

Next, the operation peculiar to the present invention which is remarkably observed in the second embodiment will be described.

When fresh blood within 6 hours after blood collection was measured under the conditions of Example 2, a two-dimensional distribution map as shown in FIG. 17 was obtained. In FIG. 17, white blood cells are hardly distributed in the area A. However, when the same sample was left for 22 hours after blood collection under the same conditions and then measured,
As shown in FIG. 8, an unknown population is seen in the area A. This unknown population is presumed to be neutrophil dead cells as described below.

First, the blood left for 22 hours after the above blood collection was treated with 1 ppm, 2 ppm, 5 ppm and 1 ppm of fluorescein diacetate (FDA) capable of staining only living cells.
Add 0 ppm and 20 ppm. The two-dimensional distribution charts at that time are shown in FIGS. 19 to 23, respectively. In these figures, it can be seen that the unknown population in region A does not move even when FDA is added, and only other white blood cells move (stained with FDA). Therefore, this unknown population is dead cells.

Next, FIG. 24 shows changes in the number of neutrophils of a sample (sample 1) and the number of particles contained in the unknown population with respect to the sample standing time. The percentage of neutrophils indicated by ● in the figure decreases with the time of leaving the sample, and conversely the percentage of the number in the unknown population increases. When the neutrophil ratio and the unknown population ratio are added, it is almost constant even if the sample is left as it is, as indicated by the circle in the figure. Other samples Also, the ratio of the neutrophil ratio and the unknown population ratio is almost constant. From the above, the unknown population is presumed to be neutrophil dead cells.

Conventionally, such dead cells, which were observed when a sample was left for a long time, could not be counted by flow cytometry, but in the present invention, the number of cells fractionated in the region A and the natural neutrophils are good. The original number of neutrophils can be obtained by adding the number of fractions at the position of the sphere.

Since it is not preferable to leave the sample for a long time in the measurement of blood items other than white blood cells, if a predetermined number or more of cells are counted in the region A, It is desirable to automatically output an alarm of leaving for a long time to call attention to the measurer.

In all the examples described above, the measurement is started after the completion of the staining (that is, after the staining reaches the equilibrium state). The staining level does not change, and the staining level of a sample with extremely large or small white blood cells always reaches an appropriate intensity in a certain period of time. Therefore, stable measurement is possible, and a signal with sufficient fluorescence intensity can be obtained even if a light source having a relatively low output is used. For example, in this example, all 10 mW argon ion lasers are used as the light source for the flow cytometer.

However, the light source of the flow cytometer used in the present invention is not limited to the above-mentioned low-power argon ion laser, but other light sources such as a mercury arc lamp, a xenon arc lamp, a He-Cd laser, and a He laser.
A light source such as a Ne laser, a krypton ion laser, or a high-power argon ion laser may be used. At that time, the dyeing condition and the measuring condition may be set according to each light source.

Now, an experiment was conducted to show the correlation between the two-step method of the present invention and the conventional parallax method, and the results are shown in FIGS. 25 to 29.

FIG. 25 shows the correlation between the result of the two-step method in which the ratio (%) of the number of lymphocytes in the total number of white blood cells is measured by fluorescence and side scattered light, and the calculation method.

The horizontal axis (X axis) shows the lymphocyte ratio by the visual calculation method, and the vertical axis (Y axis) shows the lymphocyte ratio by the two-step method. The straight line in the figure is a regression line, and the equation is a regression equation of X with respect to Y. Further, γ indicates a correlation coefficient, and n indicates the number of samples. 26 to 29 show monocytes, neutrophils, eosinophils,
The basophil ratio is shown in the same manner as in FIG. 25.

[0091]

EFFECT OF THE INVENTION Blood is measured by the reagent of the present invention,
By classifying and counting white blood cells, the following effects can be obtained.

(1) One-step dyeing in which anticoagulated blood is added to the staining solution, or two steps in which the first solution is added to the anticoagulated blood and then the second solution is added Since the sample for measurement can be obtained only by dyeing, pretreatment of the sample is easy.

(2) Since it is possible to perform the measurement in a sample preparation time of about 1 minute, the time required for the measurement can be short.

(3) Since the measurement is performed in a state where the staining is completely completed, there is no change with time of the sample during measurement, and not only a normal sample but also a sample with extremely large or small white blood cells Even at a certain time, the dyeing is always done at an appropriate intensity. Therefore, it is not necessary to change the staining time depending on the sample.

(4) Since the measurement is carried out after the dyeing is completely completed and the strong dyeing strength is reached, the light source may have a low output. Furthermore, since only one light source is required and the measurement parameters only need to measure and analyze one channel of fluorescence and one channel of side scattered light, the apparatus for carrying out the method of the present invention has a simple structure and low cost. It comes at a price.

(5) In the two-step method, only red blood cells are hemolyzed by the acid hypotonic treatment, so that simultaneous passage of red blood cells and white blood cells is eliminated, so that lymphocytes, monocytes and neutrophils due to side scattered light signals are eliminated. The separation from and became significantly better.

(6) Since the white blood cells and other blood cells are separated by the fluorescence intensity, even if not all the red blood cells are fragmented in the two-step method, the measurement value is not affected.

(7) Since the dead cells of neutrophils can be counted, the number of neutrophils can be correctly calculated even in blood left for a long time after blood collection.

(8) The dyeing process is simple because a dye (Astrazone Yellow 3G) that can dye both basophils and eosinophils is used.

In the method of the present invention, 10
When more than 000 white blood cells are measured, a measurement value excellent in accuracy and reproducibility can be obtained.

[Brief description of drawings]

FIG. 1 is a schematic view showing a specific example of an optical system of a flow cytometer used in the present invention. The symbols in FIG. 1 are explained as follows: 10 Optical system of flow cytometer 12 Laser 14 Flow cell 16 Cylindrical lens 18 Condenser lens 20 Aperture 22 Dichroic mirror 24 Side scattered light 26 Fluorescence 28 Photomultiplier tube 30 Dichroic Mirror 32 Red fluorescence 34 Color filter 36 Photomultiplier tube 38 Green fluorescence 40 Color filter 42 Photomultiplier tube

FIG. 2 is a graph showing the relative intensity of side scattered light of each white blood cell.

FIG. 3 is a diagram showing a frequency distribution of relative intensity of side scattered light when there is an influence of simultaneous passage of red blood cells.

FIG. 4 is a diagram showing a frequency distribution of relative intensity of side scattered light when there is no influence of simultaneous passage of red blood cells.

FIG. 5 is a graph showing excitation / fluorescence characteristics of Astrazone Yellow 3G.

FIG. 6 is a two-dimensional distribution diagram by fluorescence and side scattered light when stained with Astrazone Yellow 3G. In the figure, a is basophil, and b is other white blood cell.

FIG. 7 is a two-dimensional distribution map by fluorescence and side scattered light when stained with Astrazone Yellow 3G. In the figure, a is basophil, b is other leukocyte, c is eosinophil at Astrazone Yellow 3G 400 ppm, and d is eosinophil at Astrazone Yellow 3G 200 ppm.

FIG. 8 is a graph showing the excitation / fluorescence characteristics of Astrazone Orange R.

FIG. 9 is a two-dimensional distribution chart when white blood cells are classified into five using fluorescence and side scattered light. In the drawings, reference numeral 1 represents lymphocytes, 2 monocytes, 3 neutrophils, 4 eosinophils, 5 basophils, and 6 noise.

FIG. 10 is a graph showing the dependency of eosinophil and basophil fluorescence intensity on the concentration of Astrazone Yellow 3G. The symbols in the figure have the following meanings.

FIG. 11 is a graph showing the dependence of fluorescence intensity of lymphocytes, neutrophils, and monocytes on the concentration of Astrazone Yellow 3G. The symbols in the figure are as follows.

FIG. 12 is a graph showing the dependency of lymphocyte / neutrophil fluorescence intensity on the concentration of Astrazone Orange R (concentration of Astrazone Yellow 3G of 300 ppm).

FIG. 13 is a graph showing the dependence of the fluorescence intensity ratio of basophils on neutrophils on pH. The symbols in the figure have the following meanings:

FIG. 14 is a graph showing the dependence of the fluorescence intensity ratio of eosinophils on neutrophils on pH. The meanings of the symbols in the figure are the same as in FIG.

FIG. 15 is a two-dimensional distribution chart showing the results of the example of the one-step method.

FIG. 16 is a two-dimensional distribution chart showing the results of the example of the two-step method.

FIG. 17 shows 6 after blood collection in an example of the two-step method.
A two-dimensional distribution map showing the results of fresh blood within the time.

[Fig. 18] Fig. 18 shows 2 after blood collection in an example of the two-step method.
A two-dimensional distribution chart showing the results when measuring blood left for 2 hours.

FIG. 19 is a two-dimensional distribution chart showing the results when 1 ppm of FDA was added.

FIG. 20 is a two-dimensional distribution chart showing the results when FDA is added at 2 ppm.

FIG. 21 is a two-dimensional distribution chart showing the results when FDA was added at 5 ppm.

FIG. 22 is a two-dimensional distribution chart showing the results when FDA was added at 10 ppm.

FIG. 23 is a two-dimensional distribution chart showing the results when FDA was added at 20 ppm. In FIGS. 15 to 23, FL indicates the fluorescence intensity and Side Sc indicates the side scattered light relative intensity.

FIG. 24 is a graph showing changes in [neutrophil + unknown population] ratio and [neutrophil] / [unknown population] ratio depending on the sample standing time. [Neutrophils], [unknown population] ratio is sample 1
Showed only.

FIG. 25 shows the correlation between the result of the two-step method in which the ratio (%) of the number of lymphocytes in the total number of white blood cells is measured by fluorescence and side scattered light, and the visual calculation method. is there. The horizontal axis (X axis) shows the lymphocyte ratio by the visual calculation method, and the vertical axis (Y axis) shows the lymphocyte ratio by the two-step method.
The straight line in the figure is a regression line, and the equation is a regression equation of X with respect to Y. Further, γ indicates a correlation coefficient, and n indicates the number of samples.

FIG. 26 is a view similar to FIG. 25 showing the monocyte ratio.

FIG. 27 is a view similar to FIG. 25, showing the neutrophil ratio.

FIG. 28 is a view similar to FIG. 25 showing the eosinophil ratio.

FIG. 29 shows the basophil ratio similarly to FIG. 25.

Claims (6)

[Claims] 1. A dye which selectively fluorescently stains both eosinophils and basophils, a dye which fluorescently stains nuclei of white blood cells, and p.
A leukocyte five-class reagent for flow cytometers, which comprises a buffering agent for maintaining H at 6.0 to 11.0 and an osmotic pressure compensating agent for adjusting the staining solution to an osmotic pressure that maintains the morphology of leukocytes. 2. The reagent according to claim 1, wherein the dye that selectively fluorescently stains eosinophils and basophils is Astrazone Yellow 3G. 3. The reagent according to claim 1, wherein the dye that fluorescently stains the nucleus of white blood cells is one of the dyes shown below: Acridine Red Rhodamine S Rhodamine 6G Rhodamine B Rhodamine 19 Perchlorate Rhodamine 123 Eosin Y Cyanocin Cresyl First Violet Darrow Red Acrinol Phloxine FFS 1,1'-Dimethylthiocarbocyanine 1,1'-Diethylthiocarbocyanine 1,1'-Diethyl-9-methylthiocarbocyanine bromide 2- [γ- (1'-ethyl -4 ', 5'-Benzothiazolilidene) propenyl] -1-ethyl-4,5-benzoxazolidium iodide Astrazone Red 6B Basic Violet 16 2- (p-dimethylaminostyryl) -1-ethyl-
4,5-Benzothiazolium iodide 2,4-bis (p-dimethylaminostyryl) -1-ethyl-pyridinium iodide 2,6-bis (p-dimethylaminostyryl) -1-ethyl-pyridinium iodide TA-2 Astrazone Orange R. 4. A dye that selectively stains both eosinophils and basophils with fluorescence, a dye that stains leukocyte nuclei with fluorescence, and p.
A hypotonic first liquid consisting of a buffer for maintaining H in an acidic range, a buffer for neutralizing the acid in the buffer of the first liquid and maintaining the solution pH at a dyeing pH, and a solution A second liquid consisting of an osmotic pressure compensating agent for adjusting the osmotic pressure to maintain the morphology of white blood cells, and a white blood cell 5-class reagent for flow cytometer. 5. The reagent according to claim 4, wherein the dye that selectively fluorescently stains eosinophils and basophils is Astrazone Yellow 3G. 6. The reagent according to claim 4, wherein the dye that stains leukocyte nuclei fluorescently is one of the following dyes: Acridine Red Rhodamine S Rhodamine 6G Rhodamine B Rhodamine 19 Perchlorate Rhodamine 123 Eosin Y Cyanocin Cresyl First Violet Darrow Red Acrinol Phloxine FFS 1,1'-Dimethylthiocarbocyanine 1,1'-Diethylthiocarbocyanine 1,1'-Diethyl-9-methylthiocarbocyanine bromide 2- [γ- (1'-ethyl -4 ', 5'-Benzothiazolilidene) propenyl] -1-ethyl-4,5-benzoxazolidium iodide Astrazone Red 6B Basic Violet 16 2- (p-dimethylaminostyryl) -1-ethyl-
4,5-Benzothiazolium iodide 2,4-bis (p-dimethylaminostyryl) -1-ethyl-pyridinium iodide 2,6-bis (p-dimethylaminostyryl) -1-ethyl-pyridinium iodide TA-2 Astrazone Orange R.