JPH0650310B2 - Method for classifying white blood cells by flow cytometry - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for classifying blood cells in the field of clinical examination and a reagent used therefor. More specifically, it relates to a fluorescent method using a flow cytometer. The present invention relates to two methods for optically measuring stained blood cells and classifying white blood cells.

(Prior Art) The white blood cells in the peripheral blood of healthy people include lymphocytes, monocytes, neutrophils, eosinophils, and basophils. These have different functions, and by counting the white blood cells in the blood by type, they can contribute to the 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.

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 cells are smeared on a slide glass, the blood cells are fixed, further stained, and then observed under a microscope.The morphological characteristics of each white blood cell (white blood cell size, nuclear morphology,
Based on the cytoplasmic morphology, the presence or absence of granules, etc.) and the degree of staining, it is determined which blood cell the operator is and classifies and counts. At this time, a general laboratory counts 100 to 200 white blood cells, and uses the percentage (%) of each blood cell in the total number of white blood cells as a measurement value.

This method requires complicated sample preparation operations such as blood smearing, fixing, and staining before observation with a microscope,
Classification-counting with a microscope has been a great burden on the measurer due to the fact that slight differences in blood cells have to be discerned. Furthermore, 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, and at the present time, there are roughly two types of methods implemented.

One of them 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 a method that is similar in principle to the conventional disparity method, but there are many blood cells that cannot be classified by the processing by the computer, and it cannot completely replace the disparity method. There is also a problem that the device becomes complicated and large, and the price becomes high.

Another method of 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, flow an electrolytic solution in which only white blood cells float into the pores, detect impedance changes in the pores when blood cells pass through the pores, and It classifies white blood cells according to their size.

The second method comprises a light source, a flow cell in which cells in a sample flow in a narrow channel one by one, a photometric unit for detecting light emitted from the cells, and an analyzer for analyzing the detection signal. The flow cytometer provided is used. In this method, blood cells are stained, and the stained blood cells are illuminated with light, at which time the fluorescent light and possibly scattered light emitted from the blood cells are detected together, and the white blood cells are classified according to the intensity of the detected signal. Is what you are trying to do.

The method belonging to the second method is, for example, Japanese Patent Publication Sho 5
9-853 Publication and L. A. Kamensky (L.
A.Kamentsky) "Blood Cells",
Volume 6, pages 121-140, 1980. This method involves adding 10 times the amount of acridine orange dyeing solution to blood and incubating for 1 minute.
It measures the green and red fluorescence emitted from blood cells when irradiated with a light source such as an argon ion laser, and classifies and counts white blood cells based on the two-dimensional distribution.

Another example belonging to the second method is Japanese Patent Laid-Open No. 50-20.
No. 820 Publication and HMShapiro (HMShap
iro) and others "The Journal of Histo Chemistry and Sight Chemistry (The Journal of Histo
Chemistry and Cytochemistry) Vol. 24, No. 1, 39
6-411, 1976; also Vol. 25, No. 8, 9
76 to 989, 1977.
In this method, 4 times amount of Staining Solution 1 is added to blood, incubated for 3 minutes, 20% formaldehyde in the same volume as blood is added, fixed for 5 minutes, and diluted with Staining Solution II for 15 to 20 times. It is diluted with and measured with a flow cytometer. The flow cytometer used for the measurement is equipped with a mercury arc lamp that divides light into three or three lasers as a light source, each of which excites three types of fluorescent dyes contained in the dyeing solution, and the three types of fluorescent dyes. It is a device for classifying and counting white blood cells by measuring six parameters of forward scattered light, side scattered light and light absorption, and performing four-step two-dimensional distribution analysis.

Furthermore, Japanese Patent Application No. Sho 61-213 filed on Sep. 10, 1986.
No. 715 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, but unlysed erythrocytes affect measurement data and Could be unclear.

(Problems to be Solved by the Invention) In the first method of classifying and counting white blood cells by using a flow system, erythrocytes must be destroyed. In some cases, it cannot be performed completely, and there is a problem in that the accuracy of the measured value is impaired.

Japanese Patent Publication No. Sho 5 of the second method using a flow system
The method described in 9-853, etc. is characterized by measuring before the dye absorption by cells reaches equilibrium, that is, at the time when the difference in the fluorescence intensity of each leukocyte becomes maximum during the staining. I am trying. 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 differ from that of normal samples,
An appropriate staining time must be selected for each sample.
In addition, since this method attempts to classify white blood cells based only on 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 using the flow system, that is, the method described in Japanese Patent Laid-Open No. 50-20820, has many operating procedures, takes a long staining time, and uses a complicated reagent. Must. 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.

Furthermore, in the method of classifying white blood cells by flow cytometry including the invention of Japanese Patent Application No. 61-2313715, when a neutrophil becomes a dead cell because blood is left for a long time, a flow cytometer There was a problem that it could not be detected. 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 and method for accurately classifying and counting leukocytes with a simple procedure and configuration.

(Means and Actions for Solving Problems) There are two methods for classifying leukocytes of the present invention, which will be hereinafter referred to as a one-step method and a two-step method, respectively.

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

(a) A dye that selectively fluorescently stains both eosinophils and basophils, a dye that fluorescently stains nuclei of white blood cells, and a pH of 6.
Add anticoagulated blood to a staining solution consisting of a buffering agent for maintaining 0 to 11.0 and an osmotic pressure compensating agent for adjusting the staining solution to an osmotic pressure that maintains the morphology of white blood cells. ,
A step of preparing a measurement sample by staining until an equilibrium state is reached.

(b) flowing the measurement sample in a flow cytometer,
A step of distinguishing white blood cells from other blood cells based on fluorescence intensity, and measuring a lateral (90 °) scattered light signal of white blood cells and a fluorescence signal.

(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) Consists of a dye that selectively fluorescently stains both eosinophils and basophils, a dye that fluorescently stains at least lymphocytes of white blood cells, and a buffering agent that keeps pH in an acidic range A step of hemolyzing red blood cells by adding anticoagulated blood to the hypotonic first liquid.

(b) a buffer for neutralizing the acid in the buffer of the first liquid and maintaining the solution pH at the staining pH, and an osmotic pressure compensating agent for adjusting the solution to an osmotic pressure that maintains the morphology of leukocytes. The step of adding the second liquid consisting of the above to the blood sample obtained in the above (a) and treated with the first liquid to stain leukocytes.

(c) Flowing the stained sample in a flow cytometer, distinguishing leukocytes from other blood cells and ghosts by fluorescence intensity, and measuring a leukocyte fluorescence signal and a side (90 °) scattered light signal.

(d) A step of discriminating and counting the type of each white blood cell based on the fluorescence signal and the side scattered light signal emitted from the white blood cell, and calculating the ratio of each white blood cell.

In addition, white blood cells 5 of the present invention by flow cytometry
The reagents used for classification have the following composition.

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

Acridine Red Rhodamine S Rhodamine 6G Rhodamine B Rhodamine 19 Perchlorate Rhodamine 123 Eosin Y Cyanocin Cresyl Farst Biolett Darrow Lettuce Acrinor 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-benzoxazolium 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 Institute of Photosensitive Dyes: Okayama City) Astrazone Orange R (Astrazone Orange R is particularly suitable.) Now, of the fluorescence signals and side scattered light signals emitted from white blood cells, the fluorescence The signal reflects the cytochemical characteristics, and the interaction between the dye and each leukocyte gives each leukocyte a signal of different intensity.

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 few or no granules inside, and thus the scattered light intensity is the smallest, and neutrophils have many granules inside and have many nuclei. Therefore, the scattered light intensity increases. 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 classifies and counts only leukocytes in blood by a flow cytometer with only one or two steps of simple staining. Is.

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. First
The figure 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 the laser light, the white blood cells emit scattered light and fluorescence.

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

The dichroic quiller 22 reflects the side scattered light 24,
Fluorescence 2 is transmitted. The side scattered light 24 reflected by the dichroic mirror 22 is measured by the photomultiplier tube 28. Of the fluorescence 26 transmitted through the dichroic mirror 22, the red fluorescence 32 is reflected by the dichroic mirror 30 and only the green fluorescence 38 is transmitted. The reflected red fluorescent light 32 passes through a color filter 34 and is then measured by a photomultiplier tube 36. The transmitted green fluorescence 38 passes through a color filter 40 and is then measured by a photomultiplier tube 42.

By the way, the red blood cells in the measurement sample emit only very weak fluorescence, so 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 will not be measured. Does not interfere. 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, it is possible to measure fluorescence and scattered light at the same time, and only white blood cells that emit fluorescence of a certain level or more can be taken as white blood cells, but when white blood cells and red blood cells pass at the same time, red blood cells are scattered by the white blood cells. Since the scattered light due to 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, when the intensity distribution of the side-scattered light signal of the remaining white blood cells, that is, lymphocytes, monocytes, and neutrophils after the eosinophils and basophils are excluded by the fluorescence intensity is depicted in FIG. As shown in, the three populations are not completely separated.

Side scatter by lymphocytes, monocytes, and neutrophils can be achieved by further increasing the dilution factor of the measurement sample and suppressing the probability of simultaneous passage of red blood cells and white blood cells to such a degree that interference with red blood cells can be completely ignored. The frequency distribution of the optical signal intensity is as shown in FIG. 4, and these three can be completely separated. However, in order to ensure the accuracy of the measured value, the white blood cell count is about 10,
Since it is necessary to measure 000 pieces, if the dilution ratio is increased to make the sample thin, it takes a long measurement time, which is not practical.

The above-mentioned problem of interference by erythrocytes can be solved by performing erythrocyte removal operation such as erythrocyte hemolysis treatment on the measurement sample, but in the prior art, there was no erythrocyte removal method such as erythrocyte hemolysis method suitable for staining conditions. I couldn't do it.
There is no prior art example in which hemolysis is performed by classifying white blood cells into 5 by fluorescent staining. Further, there has been no method that hemolyzes only red blood cells within 1 minute and does not impair 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 Above ( A) to (e) will be described below.

a) Surfactant treatment causes morphological changes such as naked nucleation, swelling, and contraction of leukocytes at the same time as hemolysis of red leukocytes, which inhibits staining, due to scattered light signals. There is a problem that the leukocyte morphology, which makes the leukocyte fraction difficult, changes over time.

b) Ammonium salt treatment There is a problem that it inhibits staining and has a low ability to lyse erythrocytes. For example, a concentrated sample prepared by diluting whole blood by 20 times is difficult to prepare, and erythrocyte lysis takes time (3 to 5 minutes).

c) Hypotonic treatment Generally, in a hypotonic solution, due to the higher resistance of white blood cells to red blood cells, only red blood cells are hemolyzed and only white blood cells are left, but red blood cells are completely hemolyzed under physiological pH. Under the conditions, the white blood cells are also partially destroyed.

d) and e) Centrifugal separation and sedimentation separation-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, the above-mentioned simultaneous passage of red blood cells and white blood cells is used. In order to reduce the disturbance of the side scattered light intensity distribution, the red blood cells in the sample are destroyed by acid hypotonic treatment.

As described above, when hypotonic treatment is carried out in the physiological pH range, erythrocyte destruction and partial leukocyte destruction also occur.

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.

The mechanism of action of red blood cell selective hemolysis is unknown, but probably
Due to hypotonic treatment. With the progress of red blood cell hemolysis, weakening of red blood cells and acid fixation of white blood cells due to acidic pH progress,
Only white blood cells that are more resistant than red blood cells are considered to remain.

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

However, in the acidic hypotonic treatment, erythrocytes are not all fragmented, so it is difficult to completely discriminate erythrocytes from leukocytes only by the scattered light signal intensity.

Therefore, it is desirable to discriminate between red blood cells and white blood cells based on the fluorescence signal intensity as described above.

Next, the function of the dye will be described.

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

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.

Drawing a two-dimensional distribution map of the fluorescence intensity and the side scattered light intensity shows that Astrazone Yellow 3G has a low concentration (50 to 100).
ppm), only basophils are in the measurable range as shown in FIG. The fluorescence intensity of basophils becomes a constant value at 100 to 200 ppm, and does not increase above that value.

Astrazone Yellow 3G Concentration of 100-200ppm, eosinophils can be measured, and as the dye concentration increases,
As shown in the figure, the fluorescence intensity increases.

As shown in FIG. 7, since Astrazone Yellow 3G alone cannot stain lymphocytes without granules and cannot stain lymphocytes, monocytes, and neutrophils, total leukocytes cannot be measured. It is necessary to add a dye that stains the nuclei of all white blood cells without impairing the staining performance of Yellow 3G. The above-mentioned dyes that classify white blood cells into lymphocytes, monocytes, and granulocytes all meet this purpose, but in particular,
When an argon ion laser is used as the light source 12 of the flow cytometer, a dye having an excitation maximum near 488 nm is preferable. The dye thus selected is Astrazone Orange R.

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

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 map by fluorescence and side scattered light when white blood cells were classified using a dye in which Astrazone Yellow 3G and Astrazone Orange R were combined. In FIG. 9, FL is the relative intensity of fluorescence, Side Sc is the relative intensity of side scattered light, 1 is a lymphocyte, 2 is a monocyte,
3 shows neutrophils, 4 shows eosinophils, and 5 shows each group of basophils.
6 represents a noise component (hereinafter the same).

As shown in FIG. 9, white blood cells can be classified into 5 categories based on the fluorescence intensity and the side scattered light intensity. As described above, when a dye in which Astrazone Yellow 3G and Astrazone Orange R are combined is used, only one channel needs to be measured as a fluorescence signal, and therefore, in the optical system of the flow cytometer in FIG. , The dichroic mirror 30, the color filter 34, and the photomultiplier tube 36 are no longer needed.
The configuration is simple as compared with the device used in the specific example of No. 213715.

Next, the composition, pH, and osmotic pressure of the solution will be described in detail regarding the 1-step method and the 2-step method.

1 step method (a) Dye concentration Astrazone Yellow 3G concentration 5 levels of 50 to 400 ppm and Astrazone Orange R concentration of 4 levels of 100 to 400 ppm are combined to obtain eosinophils and basophils at 20 dye concentrations. The relative fluorescence intensity is shown in FIGS. 10 and 11. 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 Figure 10, Astrazone Yellow 3G density 100
At ppm or higher, the intensity of basophils increased more than that of other leukocytes, and at 200 ppm or higher, the intensity of eosinophils increased. Also, from FIG. 11, the intensity of other leukocytes was the same as that of Astrazone Yellow 3G concentration. It can be seen that the increase hardly increases. Therefore, in order to separate basophils and eosinophils from other white blood cells by the fluorescence intensity, the Astrazone Yellow 3G concentration was set to 150 to 20%.
It can be seen that it should be 0 ppm or more.

FIG. 12 shows the dependence of the fluorescence intensity of lymphocytes / neutrophils and the intensity of noise on the Astrazone Orange R concentration at an Astrazone Yellow 3G concentration of 300 ppm. Astrazone Orange R lymphocytes with a concentration of 100 ppm or more
The neutrophil fluorescence intensity and noise can now be separated,
Separation becomes good at 200 ppm or more. Above 300 ppm, the separation between lymphocytes / neutrophils and noise was almost constant, and with the increase in Astrazone Orange R concentration,
Since the fluorescence intensity of basophils and eosinophils is slightly reduced,
The Astrazone 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, respectively. It is shown in FIGS. 13 and 14. From the figure, it can be seen that the highest fluorescence intensity ratio of basophils and eosinophils to neutrophils is pH 8.5 to 9.0.

(c) Kind of Buffering Agent As the buffering agent, glycylglycine, taurine, boric acid, trycin, etc. can be used, but among them, trycin 10 to 100 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).

Two-step method Regarding the 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) pH of the first liquid The pH of the first liquid is preferably 5.0 or less for hemolyzing red blood cells, but 3.5 or more is necessary for preventing aggregation of platelets. PH 4.5 is particularly suitable.

(b) First Solution Buffering Agent As the first solution buffering agent, any buffering agent having a pKa of around 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, hemolysis of red blood cells is poor. 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 mOsm / k
g).

(d) Second solution buffering agent As the second solution buffering agent, a buffering agent having a pKa of about 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 50m
Below M, 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 Staining method 1 part by volume of EDTA2K Anticoagulated blood is added with 20 parts by volume of a staining solution, and after stirring, incubated at 25 ° C. for about 1 minute.

Selection of filter and dichroic mirror Dichroic mirror 22 510nm (510
The color filter 40 520 nm (52
(Transmitting wavelengths of 0 nm or more) Analytical results Under the above conditions, a two-dimensional distribution map based on the fluorescence intensity and the side scattered light intensity was measured with a flow cytometer.
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 liquid Second liquid Tricine sodium hydroxide (buffer) 300
mM sodium chloride (osmotic pressure adjusting agent) 750 mM pH 9.8 to 9.9 Osmotic pressure 2200 mOsm / kg Staining method 1 part by volume of EDTA2K 18 parts by volume of the first liquid was added to the anticoagulated blood, and after stirring, at 20 ° C. at 20 ° C. After incubating for 2 seconds, add 2 parts by volume of the second liquid and stir at 25 ° C for 40 seconds.
Incubate for 2 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 conditions, a two-dimensional distribution map based on the fluorescence intensity and the side scattered light intensity was measured with a flow cytometer.
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 unique action of the present invention, which is conspicuously seen in the second embodiment, will be described.

When fresh blood within 6 hours after blood collection is measured under the conditions of Example 2, a two-dimensional distribution map as shown in FIG. 17 is 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 voting under the same conditions and then measured, as shown in FIG. 18, an unknown population is seen in the region A. This unknown population is presumed to be neutrophil dead cells as described below.

First, 1 ppm, 2 ppm, 5 ppm, 10 ppm, 20 ppm of fluorescein diacetate (FDA) capable of staining only living cells is added to the blood left for 22 hours after the blood collection. The two-dimensional distribution charts at that time are shown in FIGS. 19 to 23, respectively. In these figures, even if FDA is added, area A
It can be seen that the unknown population inside does not move 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 certain 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. Adding the neutrophil ratio and the unknown population ratio,
As indicated by the circles in the figure, it is almost constant even if the sample is left unattended. 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.

In the past, 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 original position of neutrophils. The original number of neutrophils can be obtained by adding the number of fractions to

It should be noted that leaving the sample for a long time is not preferable in the measurement of blood items other than white blood cells, so
When a predetermined number or more of cells are counted in the region A, it is desirable to automatically output an alarm that the sample is left for a long time to alert the measurer.

In all the examples described above, the measurement is started after the staining is completely completed (that is, after the staining reaches the equilibrium state), so that the sample may change with time during the measurement. In addition, the staining level of a sample with extremely high or low 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-Ne laser. A light source such as 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.

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

FIG. 25 shows the correlation between the result of the 2-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. FIGS. 26 to 29 show monocyte, neutrophil, eosinophil, and basophil ratios, respectively, similarly to FIG. 25.

(Effects of the Invention) Blood is measured and white blood cells are classified and classified by the method of the present invention.
By counting, the following effects can be obtained.

One-step dyeing by adding anticoagulated blood to the staining solution, or (1) Only two-step dyeing by adding the first liquid and then the second liquid to the anticoagulated blood. Since the measurement sample is obtained, the pretreatment of the sample is easy.

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

(3) Since the measurement is performed in the state where the staining is completely completed, there is no change with time in the sample during the measurement. Further, not only a normal sample, but also a sample having an extremely large amount of white blood cells or a small amount of white blood cells is always stained with an appropriate intensity in a fixed time.
Therefore, there is no need to change the staining time depending on the sample.

(4) Since the measurement is performed after the dyeing is completely completed and the strong dyeing intensity is reached, the light source may have a low output. Furthermore, since only one light source is required, and the measurement parameters are only one channel of fluorescence and one channel of side scattered light, and the analysis is sufficient, the device for carrying out the method of the present invention has a simple structure. It will be a low price.

(5) In the 2-step method, since only red blood cells are hemolyzed by the acidic hypotonic treatment, simultaneous passage of red blood cells and white blood cells is eliminated, and therefore, lymphocytes, monocytes, and neutrophils due to side scattered light signals are separated. The separation was significantly improved.

(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 2-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, when 10000 or more leukocytes are measured per sample, a measurement value having excellent accuracy and reproducibility can be obtained.

[Brief description of drawings]

FIG. 1 is a schematic diagram 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 Light mirror 24 …… Side scattered light 26 …… Fluorescent light 28 …… Photomultiplier tube 30 …… Dichroic mirror 32 …… Red fluorescent light 34 …… Color filter 36 …… Photomultiplier tube 38 …… Green fluorescent light Light 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 side scattered light relative intensities when there is an influence of simultaneous passage of red blood cells. FIG. 4 is a diagram showing a frequency distribution of side scattered light relative intensities when there is no influence of simultaneous passage of red blood cells. FIG. 5 is a graph showing the excitation / fluorescence characteristics of Astrazone Yellow 3G. 6 and 7 are two-dimensional distribution charts by fluorescence and side scattered light when stained with Astrazone Yellow 3G. In the figure, a is basophil, b is other leukocytes, c is eosinophil at 400 ppm of Astrazone Yellow 3G, and d is eosinophil at 200 ppm of Astrazone Yellow 3G. 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 5 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 dependency of the fluorescence intensity of lymphocytes, neutrophils, and monocytes on the concentration of Astrazone Yellow 3G. The symbols in the figure are as follows. FIG. 12 shows the dependence of the fluorescence intensity of lymphocytes / neutrophils on the concentration of Astrazone Orange R (Astrazone Yellow 3G).
A graph showing a concentration of 300 ppm). 13 and 14 show the fluorescence intensity ratio of basophils to neutrophils or eosinophils to neutrophils, respectively.
A graph showing dependence on pH. The symbols in the figure have the following meanings: FIG. 15 is a two-dimensional distribution chart showing the results of the embodiment of the 1-step method. 16 and 17 are two-dimensional distribution charts showing the results of the embodiment of the 2-step method. 18 to 23 are two-dimensional distribution charts showing the results when FDA is added. 15 to 23, FL represents the fluorescence intensity and Side Sc represents the side scattered light relative intensity. FIG. 24 shows [neutrophil + unknown population] ratio and [neutrophil],
The graph which shows the change with the sample leaving time of the [unknown population] ratio. The ratios of [neutrophils] and [unknown population] are only for sample 1. FIG. 25 shows the correlation between the result of the two-step method of measuring the ratio (%) of the number of lymphocytes in the total number of white blood cells using fluorescence and side scattered light, and the visual calculation method. Horizontal axis (X
The vertical axis (Y-axis) indicates the lymphocyte ratio by the two-step method, and the vertical axis (Y-axis) indicates the lymphocyte ratio by the visual counting method. The straight line in the figure is a regression line, and the equation is a regression equation of X with respect to Y. Also γ
Indicates the correlation coefficient, and n indicates the number of samples. 26 to 29 show monocyte, neutrophil, eosinophil, and basophil ratios, respectively, similarly to FIG. 25.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-56-140253 (JP, A) JP-A-56-16872 (JP, A) Special table Sho-61-502280 (JP, A)

Claims (6)

[Claims] 1. A method of classifying leukocytes by flow cytometry, which comprises the following steps (a) to (c): (a) Selectively fluoresce both eosinophils and basophils. A dye that stains, a dye that stains the nuclei of white blood cells with fluorescence, and a pH of 6.
Add anticoagulated blood to a staining solution consisting of a buffering agent for maintaining 0 to 11.0 and an osmotic pressure compensating agent for adjusting the staining solution to an osmotic pressure that maintains the morphology of white blood cells. ,
A step of preparing a measurement sample by staining until an equilibrium state is reached, (b) flowing the measurement sample into a flow cytometer,
Differentiating white blood cells from other blood cells by fluorescence intensity, and measuring the side scattered light signal and fluorescent signal of white blood cells, (c) each white blood cell by the side scattered light signal and fluorescent signal emitted from white blood cells A step of determining the types of the white blood cells, counting them, and calculating the ratio of each white blood cell. 2. The method according to claim 1, wherein the dye for selectively fluorescently staining eosinophils and basophils is Astrazone Yellow 3G. 3. The method 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-benzoxazolium 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 method of classifying leukocytes by flow cytometry, which comprises the following steps (a) to (d): (a) Selectively fluoresces both eosinophils and basophils. Hemolyzing erythrocytes by adding anticoagulated blood to the hypotonic first liquid consisting of a dye that stains, a dye that stains the nuclei of white blood cells, and a buffer that keeps the pH in an acidic range. And (b) a buffer for neutralizing the acid in the buffer of the first liquid and maintaining the solution pH at the staining pH, and an osmotic pressure for adjusting the solution to an osmotic pressure that maintains the morphology of leukocytes. A step of adding a second liquid containing a compensating agent to the blood sample obtained in (a) and treated with the first liquid to stain leukocytes; (c) a flow cytometer for the stained sample. The white blood cells and other blood cells and ghosts are distinguished by the fluorescence intensity. Step of measuring and in which, the fluorescence signal and side scattered light signal emitted from (d) leukocytes process to determine the type of each white blood cell counting, and calculates the ratio of each leukocyte. 5. The method according to claim 4, wherein the dye for selectively fluorescently staining eosinophils and basophils is Astrazone Yellow 3G. 6. The method according to claim 4, 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-benzoxazolium 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.