JPS6370167A - Reagent used for classification of white corpuscle by flow sight metry - Google Patents

Abstract

PURPOSE:To achieve classification of white corpuscle by flow sight metry in a simple procedure, by employing a dye for fluorescent dyeing of acidocyte specifically and a dye for fluorescent dyeing of basocyte specifically. CONSTITUTION:Fresh blood subjected to an anti-coagulation processing is added to a dye liquid containing a dye for dyeing acidocyte specifically, for example, neutral red and a dye for dyeing basocyte specifically, for example, astrazone orange G to dye. The sample thus dyed flows to a flowcell 14 of a flow sight meter and is irradiated with a laser beam from a light source 12 and green fluorescence 38 is measured with a photoelectric multiplier tube 42, red fluorescence 32 with a photoelectric multiplier tube 36 and a side scattered light 4 with a photoelectric multiplier tube 28 respectively. Two signals from among the green fluorescence 38, red fluorescence 32 and side scattered light 24 are used to classify white corpuscle. Thus, while corpuscle can be classified into five categories in a simple procedure.

Description

Detailed Description of the Invention (Field of Industrial Application) This invention relates to a reagent used for classification and measurement of blood cells in the field of clinical testing. Related to reagents that can be used when classifying white blood cells by optically measuring processed blood cells (Prior technology) White blood cells in the peripheral blood of healthy people include phosphorocytes, monocytes, and neutrophils. There are three types of leukocytes, eosinophils, and basophils, each of which has a different function, and counting each type of white blood cells in the blood can contribute to the diagnosis of diseases.

For example, an increase in neutrophils is seen in inflammation, myocardial infarction, white meninges, etc., and a decrease in neutrophils is seen in viral diseases, androgenic anemia, agranulocytosis, etc. An increase in eosinophils is seen in parasitic diseases, hodgkin's disease, allergic diseases, etc. An increase in monocytes is seen during the convalescence period of infectious diseases and in patients with amniotic leukemia.

The best conventional method for classifying and counting white blood cells is called blood image analysis (visual counting method, manual method).

In this method, blood is smeared onto a slide glass, the blood cells are fixed, stained, and then observed with a microscope.The morphological characteristics of each white blood cell (white blood cell size, nuclear morphology,
The operator determines which type of blood cell it is based on the morphology of the cytoplasm, the presence or absence of granules, etc.) and the degree of staining, and then classifies and counts the blood cells. At this time, in a general laboratory, 100 to 200 white blood cells are counted, and the measured value is the percentage (%) of each blood cell in the total number of white blood cells.

This method requires complicated sacred tree creation operations such as blood smearing, fixation, and staining before observation using a microscope.
Classification and counting using a microscope places a heavy burden on the person performing the measurement, as it is necessary to discern slight differences in blood cells. Furthermore, when the number of white blood cells to be counted is small and the blood cells on the smear sample are unevenly distributed, it is difficult for even experienced measurement personnel to produce reproducible measurement values.

For this reason, there is a strong demand for a method that can automatically classify and count white blood cells, and at present, two types of methods have been realized.

One of these methods involves capturing images of blood cells using a video camera or the like, and classifying white blood cells through image processing using a computer. Although this method is close in principle to the conventional visual counting method, many blood cells cannot be classified by computer processing, so it has not completely replaced the visual counting method. Also, the equipment is complicated and large)
, there is also the problem of high prices.

The S 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 each blood cell is ηtit.
The sample is designed to pass through a large detector, and the white blood cells are classified by dividing the signal generated by the detector.

Methods using this flow system can be further subdivided into two methods.

The first method is to destroy red blood cells with a lysing agent, flow a 1tS solution in which only white blood cells are suspended into the pores, detect the change in impedance of the pores when the blood cells pass through the pores, and detect the detection signal. White blood cells are classified according to their size.

The second method consists of a light TJλ, a 70-cell in which each cell in the sample flows through a narrow channel, a photometry unit that detects the light emitted from the narrow channel, and a detection signal that is analyzed. This method uses a flow cytometer equipped with a decompression device. In this method, blood cells are stained, the stained blood cells are irradiated with light, the fluorescent light emitted from the blood cells and, in some cases, the scattered light are detected together, and the white blood cells are classified based on the intensity of the detected signal. It is.

Examples of methods belonging to this second method include:
Publication No. 9-853 and L.A. Cameratsky (L.
, A, Kamenteky) “Brad Sells (B
Load Cells) J, Volume 6, 121-1
40, 1980. This method involves adding 10 times the amount of acridine orange staining solution to blood, incubating it for 1 minute, and then measuring the green and red fluorescence emitted from blood cells when irradiated with a light source such as an argon ion laser. This classifies and counts white blood cells based on their dimensional distribution.

Other examples belonging to this second method include
Publication No. 20820 and H.M.
, M, 5hapiro) and others [The Jhana A/+ of
Histochemistry/Antodocyte Chemistry (T
he Journal of Histochemis
Try and Cytochemistry) J, No. 24G, No. 1, pages 396-411, 1976; This method involves adding 4 times the volume of staining solution 1 to blood, incubating for 3 minutes, then adding 20% formaldehyde in an equal volume to the blood and fixing for 5 minutes.
It is diluted 15 to 20 times with staining solution 11 for dilution and measured with a flow cytometer.

The flow cytometer used for this measurement is equipped with a mercury arc lamp or three lasers that split the light into three parts as a light source, and excites each of the three types of fluorescent dyes that are present in the staining solution. This device measures six parameters: forward scattered light, side scattered light, and light absorption, and classifies and counts white blood cells based on a four-stage two-dimensional distribution analysis.

(Problems to be Solved by the Invention) In the first method of classifying and counting white blood cells using a flow system, the red blood cells must be destroyed, but depending on the blood, the lysis of the red blood cells may not be complete. may not be possible, and in this case there is a problem that the accuracy of the measured value of K is impaired.

Tokuko Sho 5 of the second method using a flow system
The method described in Publication No. 9-853, etc. is characterized in that measurement is performed before the absorption of dye by cells reaches equilibrium, that is, during staining, at a time when the difference in fluorescence intensity of each white blood cell is at its maximum. It is said that

However, for specimens with an extremely high or low number of white blood cells, the staining time at which the staining intensity reaches an appropriate level will be different from that for normal specimens, and an appropriate staining time must be selected for each specimen. Must be. Furthermore, since this method attempts to separate white blood cells only based on the difference in fluorescence intensity, there is a problem in that the separation of lymphocytes and monocytes into equal volumes of blood cells is not necessarily good.

Among the second methods using a flow system,
The method described in Publication No. 0-20820 and the like requires many operating steps, takes a long time for staining, and requires the use of complicated reagents. In addition, three types of light sources are required, and six parameters must be measured, making the device extremely complex and expensive. Furthermore, since many parameters are measured in this way, analysis becomes complicated, and there is also the problem that a large-capacity analysis device is required.

This invention was made to solve the above-mentioned conventional problems, and it is possible to classify white blood cells by flow cytometry.
The purpose is to provide reagents that can be used to perform the method accurately with simple procedures and configurations.

(Means for Solving the Problems) The reagent used for classifying white blood cells into five types by flow cytometry according to the present invention has the following composition.

(1) A dye that specifically and strongly stains eosinophils, such as neutral red. (2) A dye that specifically and strongly stains basophils: Astrazone Orange G or Auramine 0 (Astrazone Orange G is particularly preferred). (3) Buffer (phosphoric acid,
Citric acid, boric acid, TR Engineering S, HEPIS, glycine,
carbonic acid, collidine, taurine, etc.) (4) Osmotic pressure compensator (alkali metal salt) In addition, the following component (5) may be added in order to better separate the monocyte fraction.

(5) A dye that strongly stains monocytes specifically, DloC1 (
3).

D10C2 (3), DloC3 (3), D10C5 (3
), DloC6(3), TA-2 and 1-[γ-(1
At least one of '-ethyl-47,s/-penzothiazolidene) flobenyl]-1-ethyl-4,5-benzooxacillium iosite.

(% DloC3 (31 is preferred)) the above components (1
The chemical structural formulas of the dyes used as ), (2) and (5) are as follows.

H DiOCn(3) (1,1'-dialkyloxacarbocyanine) n=1.223. 5.6 2-(r-(1'-ethyl-4',5'-benzothiazolylidene) flobenyl]-1-ethyl-4,S-benzoxacilium iosito) The reagents of this invention are It is used to classify and count only white blood cells in blood using a flow cytometer, with no processing or other operations required, and a simple one-step staining process.

A specific example of the optical system of a flow cytometer in which the reagent of the present invention is used will be explained based on the drawing shown in FIG.

FIG. 1 shows a case where side scattered light, daylight, and green fluorescence are measured. Optical system 10 of this flow cytometer
The light source used for this has a wavelength of 488 nm * lfj
Power: 10 mW argon ion laser-12. The light emitted from the laser 12 is curved by the cylindrical lens 16 and irradiates the measurement sample flowing through the flow cell 14 .

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

Among these, the scattered light and the fluorescent light emitted to the side are collected by the condenser lens 18, pass through the aperture 20, and then reach the guichroic mirror 22.

The guichroic mirror 22 reflects the 1nil scattered light 24 and transmits the fluorescent light 26. Guicroic mirror 2
The side scattered light 24 reflected by the photomultiplier tube 2
Measured by 8. Of the fluorescent light 26 transmitted through the guichroic mirror 22, daylight 32 is reflected by the guichroic mirror 30, and only green fluorescent light 38 is transmitted. The reflected daylight light 32 is passed through a color filter 3
4, it is measured by a photomultiplier tube 36. The expelled green fluorescence 38 passes through a color filter 40 and is then measured by a photomultiplier tube 42.

By the way, among the plurality of signals emitted by white blood cells,
The side-scattered light signal reflects intracellular information; in other words, the larger the pure cell nucleus in the cell and the more granules there are, the stronger the light reflection within the cell becomes, and the side-scattered light intensity increases. increase Therefore, lymphocytes have no or few granules inside, so the intensity of scattered light is the lowest, whereas neutrophils have many granules inside, and have a large nucleus, so the intensity of scattered light is the lowest. The light intensity is the highest.

The intensity of light scattered by monocytes, eosinophils, and basophils is between that of lymphocytes and neutrophils. For this reason, the relative intensity of the side scattered light of each white blood cell is as shown in FIG. 2.

On the other hand, the fluorescent signal reflects the cytochemical characteristics, and the interaction between the dye and each leukocyte results in a signal of different intensity from each leukocyte.

Therefore, by combining the side-scattered light signal with at least one fluorescent signal, depending on the dye, it becomes possible to better classify leukocytes.

Now, white blood cells are detected by 6
Dyes that can be classified into four or more types based on two-dimensional distribution based on two parameters among the 11 constant/parameters (however, the light source is an argon ion laser
It was experimentally found that there are 17 types (when limited to 483 nm). (LA, p!,) In particular, more than five classifications are possible for acridine orange, rhodorine orange, etc.

When fluorescing the 17 types of dyes mentioned above to sufficiently distinguish white blood cells from red blood cells and platelets, and using a flow cytometer to draw a two-dimensional distribution using side-scattered light and fluorescence, the results are as shown in Figures 3 to 5. A pattern can be obtained.

However, in Figures 3 to 5, 1 represents lymphocytes, 2 represents monocytes, 3 represents neutrophils, 4 represents eosinophils, and 5 represents basophils. Further, 5ide Sc represents the relative intensity of side scattered light, and 1i'L represents the relative intensity of fluorescent light.

The pattern shown in FIG.
θ), oxacarbocyanine, and acridine dyes. For acridine dyes, the two-dimensional distribution of green and red fluorescence shows a similar pattern.

The pattern shown in Figure 4 appears in neutral red.

The pattern shown in Figure 5 is Astrazone Orange G1.
Seen in auramine O.

It was also discovered that there are about 20 types of dyes that classify leukocytes into three categories, and the patterns shown in FIG. 6 can be obtained with these dyes.

By appropriately mixing two types of dyes as shown in Figures 4 and 5,
When the fluorescent light of both dyes is received, a pattern as shown in Figure 7 is obtained with the irradiated light/side scattered light.Also, the fluorescent light is divided into two colors: green fluorescent light (Green FL) and red fluorescent light ( Red
By appropriately selecting the wavelength of FL, ) according to the characteristics of the dye, eosinophils 4 and basophils 5 can be separated from other leukocytes as shown in FIG. The remaining portion can be separated by fluorescence/side scattered light as shown in Figure 9.

When a dye giving the pattern shown in FIG. 6 is added to the condition shown in FIG. 7, the separation of lymphocytes 1, monocytes 2, and neutrophils R3 becomes even better, resulting in a pattern like that shown in FIG. 10. In this case as well, green/red fluorescence (Figure 11) and fluorescence/side scattered light (Figure 12)
A two-step analysis is also possible.

Function of dye a. Neutral red leukocytes are fluorescently stained with a dye, which particularly strongly stains eosinophils 4, and the leukocytes are fractionated using scattered light and red fluorescent light as shown in Figure 4.

FIG. 13 shows the excitation and fluorescence characteristics of neutral red.

When dyed with Cono Neutral Red, special Ni580 ~
Eosinophils are strong in the 640 nm (orange-red) wavelength band (
specifically) stained.

A two-dimensional distribution as shown in Fig. 4 can be obtained with a staining solution having a pH of 5 to 11 and a dye concentration of 3 to 300μ. Even if the dye concentration is 3μ97ml or less, eosinophil-specific distribution nomiturns can be obtained, but other white blood cells are noisy and cannot be measured accurately. Signal from eosinophil 4 only! The dye concentration may be about 0.1μ9/ng or higher if the dye concentration is low.

b. Astrazone Orange G This is a dye that fluorescently stains leukocytes, particularly basophils 5, and the leukocytes are fractionated using scattered light and green fluorescent light as shown in Figure 5.

FIG. 14 shows the excitation and fluorescence characteristics of Astrazone Orange G.

When stained with this Astrazone Orange G, especially 54
Basophils are strongly (specifically) stained in the (yellow-green) fluorescent wavelength band centered around 0 nm.

A two-dimensional distribution as shown in the fifth example can be obtained with a dye solution having a pH of 5 to 11 and a dye concentration of 1 to 30011 g/particle.

Acridine Orange G TolnJ-like results are also observed with Auramine O.

C0 and other dyes A dye that fluorescently stains leukocytes, particularly strongly stains monocytes, and uses four-way scattered light and fluorescent light to divide leukocytes into at least three groups as shown in Figure 6.

d. Combination of Neutral Red and Astrazone Orange Q Neutral red strongly stains eosinophils 4, Astrazone Orange G strongly stains basophils 5, and white blood cells are stained with scattered light and yellow to red fluorescence. Fraction as shown in Figure 7.

pH5-11, neutral red concentration 01-30μ9
/yt1. 'Astrazon Orange Gp degree 1-300
A two-dimensional distribution as shown in FIG. 7 can be obtained with the μ9/wit staining solution.

e, Combination of Neutral Red, Astrazone Orange G and other dyes By appropriately combining dyes d and C, separation of monocytes 2 is better than in d (contamination of phosphorocytes 1 and neutrophils 3). This makes it possible to stain white blood cells in a state with a small amount of white blood cells, and the white blood cells are fractionated using side scattered light and yellow to red fluorescence as shown in Figure 10.

The dye C above which can be used for this purpose is DloC
l(3), D10C2(31, D10C3(3),
L)10C5(3), DtOCfi3).

Oxacarbocyanine dyes such as TA-2 (styryl dye manufactured by Japan Photosensitive Color Research Institute (Okayama City)), 2
cyanine dyes such as -[γ-(1'-ethyl-4',5'-benzothiacylyrif'n)propenyl]-1-ethyl-4,5-benzooxacillium iosito; and the like.

As shown in Figure 15, the oxacarbocyanine dye stains eosinophils 4 weakly than neutrophils 3, and separates leukocytes into four categories, but in nature, the mixed neutral red is strongly eosinophil-specific. Eosinophils 4 are distributed on top of neutrophils 3 according to the staining characteristics.

Although there are other dyes belonging to Group C, only the above-mentioned dyes and their analogs can be used for this purpose due to restrictions such as dyeing conditions, dyeing intensity, and fluorescent wavelength.

Other staining solution components a and buffer are used to maintain the staining solution at an optimum pH. Maintaining the pH is important because the staining properties of the dye and the type of white matter in blood cells to be stained differ depending on the pH. Blood itself has a pH of 7.4.
Since it has a buffering function to maintain the pH at a certain level, an amount of buffering agent is required to counteract this effect and achieve the desired pH.

For this purpose 5-200 mM phosphorus r37, citric acid, boric acid, TR-S, HEPES, glycine, charcoal-11
, collidine, taurine, etc. are used.

b. Osmotic pressure compensator: 4 of the physiological osmotic pressure of human blood (280 moθmA9), which is added to prevent extreme deformation and hemolysis of leukocytes.
60mM to 380m varying from 0% to 250%
An alkali metal salt of M is used.

When implementing this invention, the following points must be kept in mind.

a, the optimal concentration of the dye to be mixed for specific staining, p
Since H, etc. are generally different, staining conditions must be found to use all dyes with the desired specificity.

b. Each dye has a fluorescent intensity (in general, the fluorescent intensity of each dye is multiplied by the amount of irradiation, the absorbance coefficient ε at that wavelength, the quantum efficiency φ, and the correction coefficient α by the dye concentration C2 optical system. ) are different, so in order to obtain the desired pattern,
ta a of each amount of dye added? What you have to do.

The receiving wavelength kM must be selected so that a two-dimensional pattern with C5 specificity can be obtained.

(Example) The present invention will be explained in more detail with reference to Examples 1 to 6 below.

Example 1 A staining solution was prepared by adding Astranon Orange G and Neutral Red to a 10 mM boric acid buffer solution with pH 9.0 containing 75 mM Na (concentrations of Neutral Red and Astrazone Orange G) in Table 1. .

Add 80μ of EDTA anticoagulated fresh blood to 2rxl of these staining solutions.
After incubating for 1 minute, green fluorescence, daylight, and side scattering of white blood cells were measured using a flow cytometer with an optical system as shown in Figure 1.The results were as shown in Table 1. White blood cells were separated into several types.

*1) After separating eosinophils and basophils using daylight light *6)/side scattered light *3) After separating eosinophils and basophils using daylight light/green fluorescent light *7), the remainder is separated using fluorescent light/side scattered light. 5 classifications that divide into 3 parts *4) 4 classifications for daylight/side scattered light *5) 3 classifications for daylight/green fluorescence including eosinophils *2) Unclassifiable, but daylight; 580 nm or more edge Fluorescence; 520-580 nm Example 2 (pH) 75mM NaCA! Three types of staining: Astrazone Orange GIO μg/ml, Neutra/L/L/Nod 1119F10, 0/y. was prepared and measured in the same manner as in Example 1. As a result, at pH 10.0, daylight/(Ill) cannot be classified into 5 by the scattering source,
After fractionating basophils and eosinophils using repeated light/red fluorescence, the remaining three types are fractionated using repeated 71:/side scattering light, making it possible to classify all five types of white blood cells. At pH 9.0, there are five classifications of daylight/side scattered light. pHs. o, four fractions are possible with daytime light/side scattered light.

Example 3 (NaCl3 concentration) lomM borate buffer pH containing Astrazone Orange GIOμ9/rnl, Neutral Red 1μ91at
9.0 with Na (J f 5 0, 7 5, 150, 3
Four staining solutions containing 00mM were prepared and 6111 assay was performed in the same manner as in Example 1. There are no particularly large changes in the fractionation pattern between these axes, and leukocytes can be classified into five types using any staining solution.

Example 4 (buffer concentration) NaC675mM, Astrazone Orange GIO
μ9/Pay Neutral Red 1μ'a/rttl'f
r: All three types of staining solutions were prepared in which buffer concentrations of b-containing boric acid buffer solutions were 3, 10, and 30 mM at pH 9 and 0, and measurements were performed in the same manner as in Example 1. Within this range, there were no particularly large changes in the fractionation pattern, and leukocytes could be classified into five types even with every 7 L of staining solution.

Example 5 (fluorescence wavelength) 75mM NaC7, Astrazone Orange G1 0
6411 was determined in the same manner as in Example 1 using a staining solution of lQmM borate buffer pH 9.0 containing μ'a/me, neutral red 1s7mt; However, a total reflection mirror is used in the position of the microchroic mirror 30 in FIG.
nm or more, 580 nm or more, 600 nm or more, 620 nm or more
Six types of firefly ytS of nm or larger were received and analyzed using fluorescence/side scattered light.

When the light receiving wavelength is adjusted to the full-length wavelength, basophils and lymphocytes become relatively close to each other, and eosinophils and neutrophils move away from each other. The receiving wavelength that allows good separation of the five classifications is 5f30nm or more.
Two with a wavelength of 580 nm or more are particularly good.

Example 6 (Lunch light, repeated light wavelength) The same measurements as in Example 5 were carried out, and daytime light and repeated light having the wavelengths shown in Table 2 were received.

Table 2 (1 540 ~ 600 560 or more 580 or more
540-580 5 6 (1 or more d
580 or more 5()0~
Basophils and eosinophils are specifically and strongly stained with fluorescence wavelengths of 540 5 60 or higher, especially C and e, and can be easily separated from other leukocytes.

In addition, in all of the examples described above, the measurement is started after the staining is completely completed, that is, after the staining has reached an equilibrium state, so the sample does not change over time during the definition. Furthermore, even for specimens with extremely high or low white blood cells, the staining level always reaches an appropriate intensity within a certain period of time. Therefore, stable measurement is possible, and a signal with sufficient fluorescence intensity can be obtained even when a light source with relatively low ITL power is used. For example, in all of the examples, an IQmW alkon ion laser is used as the light source of the flow cytometer.

From the above examples, it was found that good results were obtained when the above reagents were used under the following conditions.

Astrazone Orange G; 3-100 (μ9//')
Neutral red; 03-10 (μ9/me)
pH; 8.0 to 11.0 Fluorescent wavelength green emission; 500 to 580 nm red fluorescence
: 560 nm or more However, the light source of the flow cytometer used in this invention is not limited to the aforementioned low-output argon ion laser, but may also include other light sources such as a mercury arc lamp, a xenon arc lamp, a He-Cd laser, A light source such as a He-Nθ laser or a high-output argon ion laser may be used. At that time, it is only necessary to set the optimum dye, dyeing conditions, and measurement conditions according to each light source.

(Effects of the Invention) When blood is measured using a flow cytometer using the reagent of the present invention, and leukocytes are classified and counted, the following effects can be obtained.

(1) Pretreatment of the sample can be made very simple because a III standard trial sample can be obtained by only one step of staining, which involves adding anticoagulated blood to the staining solution.

(2) Since it is possible to perform the measurement in a dyeing time of 1 degree, the time required for measurement can be shortened.

(3) Since 6111 is determined after staining is completely completed, there is no change in the sample over time during measurement, and it can be used not only for normal samples but also for samples with extremely high or low white blood cells. Staining is always done at the appropriate intensity within a certain period of time. Therefore, there is no need to change the staining time depending on the specimen.

(4) Since measurement is performed after staining is complete and strong staining intensity has been reached, the light source of the flow cytometer may be of low output. Furthermore, only one light source is required, and the reagent of this invention can be used because it is only necessary to select two parameters from two channels of fluorescent light and one channel of side scattering light, and then perform the analysis. The device is simple in construction and inexpensive.

(5) Since the reagent of the present invention has a very strong ability to stain cells, it is possible to obtain classification results for each leukocyte with a high degree of separation.

(6) When side scattered light is measured in addition to fluorescence, classification results for each white blood cell with even better separation, including the separation of lymphocytes and monocytes, can be obtained.

(7) Red blood cells, platelets, and immature red blood cells can be completely distinguished from white blood cells because their fluorescence intensity is much lower than that of white blood cells. Therefore, if the reagent of the present invention is used, there is no need to lyse red blood cells beforehand.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing a specific example of the optical system of a flow cytometer in which the reagent of the present invention is used. The symbols in the figure are as follows: 10: flow site optical system, 12: laser, 14:
Flow cell, 16: Flint 9 linear lens, 22: Dichroic mirror, 24: Side scattered light, 26: *Part,
28: Photomultiplier tube, 30: Dichroic mirror, 3
2: Daylight, 34: Color filter, 36: Photomultiplier tube, 38: Green emission, 40: Color filter, 42:
Photomultiplier tube, FIG. 2 is a graph showing the side scattered light intensity of each white blood cell. Figures 3 to 12 and 1512'l are two-dimensional distribution diagrams when white blood cells are classified using two signals. In these distribution maps, 1 is lymphocyte, 2 is monocyte, 3 is neutrophil,
4 represents a collection of eosinophils and 5 represents a collection of basophils. FIG. 13 is a graph showing the excitation/fluorescence characteristics of Neutral Red, and FIG. 14 is a graph showing the excitation/fluorescence characteristics of Astrazone Orange G. In each drawing, 5ide Sc is the relative intensity of side scattered light, P'
L, r<edi+'L, and Green F'L
, respectively, are the relative intensity of fluorescent light, the relative intensity of daylight, and the relationship of green luminescence.
5ide 5c. Abusive 7 concave spoon 10 figure 1215 mouth 5ide 5c. Koto Q2 Horse q concave H concave back, 12 concave RedFL, 5ide 5c. 2. Name of the invention Reagent used for white blood cell classification by flow cytometry 3. Relationship with the case of the person making the amendment Patent applicant Inl Koshirae o1 group le 9tl diagonal hira f correction insert -6,
Contents of amendment (1) The description is amended as follows. 100 lines False Correct 8 8 It's a lot, but is it a lot? 1616
Nikurisin Nikurisin 21 7 Mixed in Mixed 25 9 Kai Raiko
*6) Kairaiko 2510 Green Fluorescence
*7) Green fluorescent 31 lower 3 flow site
Flow cytometer Red FL. Procedural amendment April 2, 1986

Claims (5)

[Claims] (1) A leukocyte classification reagent for use in a flow cytometer comprising a dye that specifically fluorescently stains eosinophils and a dye that specifically fluorescently stains basophils. (2) The reagent according to claim 1, wherein the dye that specifically fluorescently stains eosinophils is neutral red. (3) The reagent according to claim 1, wherein the dye that specifically fluorescently stains basophils is any one of a, astrazone orange Gb, and auramine O. (4) A flow cytometer comprising a dye that specifically fluorescently stains eosinophils, a dye that specifically fluorescently stains basophils, and a dye that specifically fluorescently stains monocytes. Reagent for classifying 5 leukocytes. (5) The dye that specifically fluorescently stains monocytes is 1,1'-
Dimethyloxacarbocyanine, 1,1'-diethyloxacarbocyanine, 1,1'-di-(n-propyl)
Oxacarbocyanine, 1,1'-di-(n-pentyl)oxacarbocyanine, 1,1'-di-(n-hexyl)oxacarbocyanine, TA-2 or 2-[γ-(1'-ethyl) The reagent according to claim 4, which is -4',5'-benzothiazolilidene)propenyl]-1-ethyl-4,5-benzoxazolium iodide.