JP2008111717A - Reagent for measuring reticulocyte and platelet, reagent kit for measuring reticulocyte and platelet, and measuring method of reticulocyte and platelet - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent for measuring the reticulocyte and the platelet capable of clearly discriminating the reticulocyte and the platelet from other blood corpuscle components or impurities in blood to measure them. <P>SOLUTION: The reagent for measuring the reticulocyte and the platelet contains a first coloring matter for dyeing the reticulocyte and a second coloring matter for dyeing the platelet and the second coloring matter is at least one kind of a coloring matter selected from the group consisting of capriblue, nileblue and brilliant cresylblue. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a reagent for measuring reticulocytes and platelets, a reagent kit, and a measuring method.

Human blood contains various blood cell components of red blood cells, white blood cells and platelets. Among them, platelets are anucleated cells having a diameter of 2 to 3 μm, and 150,000 to 350,000 / μl are present in normal human blood.
However, it is known that the number of platelets in a specimen decreases when suffering from thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) or a disease such as acute leukemia. In general, when the number of platelets in the blood is lower than 10,000 / μl, it is judged that there is a need for blood transfusion. Therefore, it is important in the clinical field to measure the platelet count quickly and accurately.

  Blood also includes reticulocytes. Reticulocytes are young erythrocytes immediately after erythroid cells in the bone marrow are enucleated and released into the peripheral blood. As a remnant of the cell maturation process, reticulocytes are characterized by having small amounts of RNA or organelles such as ribosomes and mitochondria that are not contained in mature erythrocytes.

  In the field of clinical examination, classifying and counting reticulocytes is extremely important for grasping the hematopoietic state in the bone marrow of a patient. In healthy people with normal bone marrow hematopoiesis, the number of reticulocytes accounts for 0.5-3.0% of all red blood cells, whereas bone marrow hematopoiesis is abnormal, for example, bone marrow hematopoiesis is suppressed. The number of reticulocytes decreases in the treated state and increases in the state where bone marrow hematopoiesis is enhanced. Specifically, it decreases at the time of chemotherapy for aplastic anemia and malignant tumor, and increases at hemolytic anemia.

  2. Description of the Related Art Conventionally, a method for quickly measuring cells in blood, that is, blood cells such as leukocytes, reticulocytes, and erythrocytes, using the principle of flow cytometry is known. As such a measuring method, for example, JP-A-10-282094 (Patent Document 1) discloses a method for counting and identifying reticulocytes, erythrocytes and platelets in a whole blood sample, and a reagent composition used in the method. It is disclosed. In the method of Patent Document 1, reticulocytes are stained with a reagent mixture containing a cationic dye, particularly oxazine 750, and scattered light and absorbed light are measured using flow cytometry, and these scattered light and fluorescence are used as parameters. Reticulocytes are counted and distinguished from red blood cells and platelets.

  Japanese Patent No. 2613250 (Patent Document 2) discloses that leukocytes in undissolved whole blood are obtained from red blood cells and platelets, including contacting a reagent containing a fluorescent dye that absorbs red light with undissolved whole blood. A method for identification is disclosed. It is described that an oxazine dye is used as a fluorescent dye that absorbs red light, and this stains leukocytes, so that leukocytes can be distinguished from erythrocytes and platelets by measurement using flow cytometry.

  Furthermore, Japanese Patent No. 3485436 (Patent Document 3) discloses a reagent for measuring reticulocytes comprising a dye that specifically stains at least one reticulocyte and a dye that specifically stains leukocytes. This patent document also describes that oxazine dyes can specifically stain leukocytes.

  In the measurement of platelets, it is known that disrupted erythrocytes, lipids, and the like appearing in blood have an influence as measurement contaminants because their sizes are similar to platelets. In particular, the measurement of a sample with a low platelet count that requires blood transfusion has a greater effect of these contaminants. However, Patent Documents 1 to 3 described above do not describe measuring reticulocytes and platelets with higher accuracy while suppressing the influence of such impurities.

Japanese Patent Laid-Open No. 10-282094 Japanese Patent No. 2613250 Japanese Patent No. 3485436

  In view of the above-mentioned present situation, the present invention can measure reticulocytes and platelets more clearly from blood contaminants such as other blood cell components and lipid particles in a measurement method using flow cytometry. An object is to provide a reagent for measuring reticulocytes and platelets.

  The present invention comprises a first dye for staining reticulocytes and a second dye for staining platelets, wherein the second dye is composed of capri blue, nile blue and brilliant cresyl blue. A reagent for measuring reticulocytes and platelets, which is at least one dye selected from the above.

  The present invention also includes a first reagent containing a first dye for staining reticulocytes and a second reagent containing a second dye for staining platelets, wherein the second dye is capri blue. And a reagent kit for measuring reticulocytes and platelets, which is at least one dye selected from the group consisting of Nile blue and Brilliant cresyl blue.

  Furthermore, the present invention provides a first reagent containing a buffer for keeping the pH constant during measurement, a first dye for staining reticulocytes, and a second dye for staining platelets. And a reagent kit for measuring reticulocytes and platelets, wherein the second dye is at least one dye selected from the group consisting of capri blue, nile blue and brilliant cresyl blue.

The present invention relates to a first dye for staining reticulocytes, a second dye for staining platelets that are at least one selected from the group consisting of capri blue, Nile blue and brilliant cresyl blue, and a specimen. To prepare a sample for measurement,
Irradiate the cells in the obtained measurement sample with light, measure the scattered light and fluorescence emitted from the cells,
It is also a reticulocyte and platelet measuring method including detecting reticulocytes and platelets based on detected scattered light and fluorescence.

Heretofore, it has not been known that a specific oxazine dye found for staining platelets can specifically stain platelets to such an extent that it can be distinguished from contaminants such as platelets and lipid particles. This oxazine dye can increase the fluorescence intensity of platelets when measured by flow cytometry by fluorescently staining platelets. Thereby, it is possible to distinguish between platelets and contaminants such as lipid particles.
In the present specification, “contaminants” in blood are components in blood that can affect the measurement of reticulocytes and platelets, and include lipid particles, disrupted erythrocytes, and the like.

  By using the reticulocyte and platelet measurement reagent of the present invention, even a sample containing contaminants that can hinder the detection of platelets, suppressing the effects of such contaminants and other blood cell components in the blood, Reticulocytes and platelets can be measured with higher accuracy and speed.

  In the measuring method using flow cytometry, the present inventors can measure reticulocytes and platelets more clearly from blood contaminants such as other blood cell components and lipid particles. As a result of repeated research using more than 100 kinds of dyes to obtain a measuring reagent, it was found that a specific oxazine dye can specifically stain platelets. Furthermore, by using a reagent that contains a dye capable of specifically staining reticulocytes together with this specific oxazine dye, reticulocytes and platelets can be more clearly distinguished from other blood cell components and blood contaminants. The present invention has been completed by finding out what can be done. The reagent for measuring reticulocytes and platelets of the present invention contains a first dye for staining reticulocytes. The first dye may be any dye that can stain reticulocytes so as to be distinguishable from other blood cells or impurities, and is preferably a nucleic acid-staining cyanine dye. As the nucleic acid-staining cyanine dye, conventionally known dyes can be used. For example, the dye disclosed in Japanese Patent No. 3485436, the dye disclosed in JP-T-2001-506686, and disclosed in JP-T-2003-532790. And dyes disclosed in US Pat. No. 4,957,870. Above all, the following formula:

Wherein R ₁ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or —CH ₂ (CHR ₅ ) _x OR ₆ ;
R ₂ and R ₃ are the same or different and are each a hydrogen atom, a halogen atom, a cyano group, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, an aryl group, or an aralkyl group;
R ₄ is an alkyl group having 1 to 6 carbon atoms, —CH ₂ (CHR ₇ ) _y OR ₈ , an aryl group or an aralkyl group;
R ₅ and R ₇ are each a hydrogen atom or a hydroxyalkyl group having 1 to 3 carbon atoms;
R ₆ and R ₈ are each a hydrogen atom, an acyl group, or an alkyl group having 1 to 3 carbon atoms;
Z is a sulfur atom, an oxygen atom, a selenium atom or CR ₉ R ₁₀ ;
R ₉ and R ₁₀ are the same or different from each other and each is an alkyl group having 1 to 3 carbon atoms;
n is an integer of 1 or 2;
x and y are each an integer of 0 to 3;
X ⁻ is an anion)
Is preferably used.

The alkyl group having 1 to 6 carbon atoms in R ₁ of the above formula (I) may be linear or branched, for example, methyl, ethyl, propyl, butyl, isobutyl, sec-butyl, Examples include tert-butyl, pentyl and hexyl groups. Of these, a methyl or ethyl group is preferable.

The groups represented by R ₂ and R ₃ in the above formula (I) may be substituted at any of the ortho, meta and para positions. Examples of the alkyl group having 1 to 6 carbon atoms in R ₂ and R ₃ are the same as those described above. Examples of the alkoxy group having 1 to 6 carbon atoms include methoxy, ethoxy and propoxy groups, and among them, methoxy or ethoxy group is preferable. Examples of the aryl group include a phenyl group. Examples of the aralkyl group include a benzyl group.
The group represented by R ₂ and R ₃ in formula (I) is more preferably hydrogen.

Examples of the alkyl group having 1 to 6 carbon atoms, the aryl group and the aralkyl group in R ₄ of the formula (I) are the same as those described above.

Examples of the hydroxyalkyl group having 1 to 3 carbon atoms in R ₅ and R ₇ include hydroxymethyl, hydroxyethyl, and hydroxypropyl groups. Among them, hydroxymethyl or hydroxyethyl groups are preferable.

The acyl group in R ₆ and R ₈ is preferably an acyl group derived from an aliphatic carboxylic acid. Such acyl groups include acetyl and propionyl groups, with acetyl groups being preferred. Examples of the alkyl group having 1 to 3 carbon atoms are the same as those described above.

Z of formula (I) above, a sulfur atom, an oxygen atom, a selenium atom or a CR ₉ R _10, among others sulfur atom is preferable.
Examples of the alkyl group having 1 to 3 carbon atoms in R ₉ and R ₁₀ are the same as those described above.

As anions in X ⁻ of the above formula (I), halogen ions (fluorine, chlorine, bromine or iodine ions), boron halide ions (BF ₄ ⁻ , BCl ₄ ⁻ , BBr ₄ ⁻ etc.), phosphorus compound ions, Examples thereof include halogen oxygenate ions, fluorosulfate ions, methylsulfate ions, and tetraphenylboron compound ions having a halogen atom or a haloalkyl group as a substituent on the phenyl ring. Of these, bromine ion or BF ₄ ⁻ is preferable.

Examples of the dye of the above formula (I) are shown below.

Among the dyes of the above formula (I), the dye of n = 1 is, for example, the formula:

Is reacted with N, N-disubstituted formamidine and the product is converted to the formula:

It can synthesize | combine by making it react with the quinoline derivative represented by these, and then processing with sodium borofluoride.
In addition, the compound of n = 2 in the above formula (I) can be prepared by, for example, malondialdehyde bis (phenylimine) instead of using N, N-disubstituted formamidine in the reaction of synthesizing the dye of n = 1. ) It can be synthesized by using a salt.

  In addition to the above-mentioned nucleic acid-staining cyanine dye, dyes such as new methylene blue and oxazine 750 are also known as dyes for reticulocytes, and these may be used as the first dye of the present invention.

  Although said 1st pigment | dye changes also with the kind of pigment | dye, it is preferable to be contained in a reagent by the density | concentration used as 0.1-50 ppm when it mixes with a test substance, More preferably, it is about 0.5-10 ppm. Within such a concentration range, reticulocytes can be distinguished from other blood cell components and impurities.

  In the composition of the reagent of the present invention, the above dye for staining reticulocytes can quickly penetrate into reticulocytes and stain intracellular RNA.

で表されるカプリブルーＧＯＮが挙げられる。ナイルブルーとしては、例えば次の式： The reagent for measuring reticulocytes and platelets of the present invention contains a second dye for staining platelets. The second dye is a dye that can specifically stain platelets and is at least one dye selected from the group consisting of capri blue, nile blue, and brilliant cresyl blue. The second dye is preferably one that can stain platelets in a distinguishable manner from lipid particles. As Capri Blue, for example, the following formula:

Capri blue GON represented by. As Nile Blue, for example, the following formula:

（式中、Ｘ^-はＣｌ^-又は１／２ＳＯ₄ ^2-を表す）
で表されるナイルブルーが挙げられる。ブリリアントクレシルブルーとしては、例えば次の式：

(Wherein, X ^- is Cl ^- represents a or 1 / 2SO ₄ ^2-)
Nile blue represented by As brilliant cresyl blue, for example, the following formula:

で表されるブリリアントクレシルブルーＡＬＤが挙げられる。これらの色素は、市販されており、例えば、カプリブルーＧＯＮはクロマ社から、ナイルブルーは東京化成、シグマ−アルドリッチ社から、ブリリアントクレシルブルーＡＬＤはシグマ−アルドリッチ社からそれぞれ入手することができる。

The brilliant cresyl blue ALD represented by these is mentioned. These dyes are commercially available. For example, Capri Blue GON can be obtained from Chroma, Nile Blue from Tokyo Kasei and Sigma-Aldrich, and Brilliant Cresyl Blue ALD from Sigma-Aldrich.

  It was also found that the dye for staining platelets can stain platelets in a distinguishable manner from crushed red blood cells.

The second dye for staining the platelets described above varies depending on the type of the dye, but is preferably contained in the reagent at a concentration of 0.01 to 10 ppm when mixed with the specimen, more preferably 0.1 About 2.0 ppm. In particular, when capriblue GON is used as a dye for staining platelets and platelets are distinguished from lipid particles or crushed erythrocytes, 0.2 to 3.0 ppm is preferable, and 0.5 to 2.0 ppm is more preferable. When discriminating platelets from lipid particles or crushed red blood cells using Nile Blue, 0.1 to 2.0 ppm is preferable. In the case where platelets are distinguished from lipid particles or crushed erythrocytes using brilliant cresyl blue ALD, 0.5 to 3.0 ppm is preferable, and 1.0 to 2.0 ppm is more preferable.
Such a concentration range is preferable because the distinction between platelets and other blood cell components and impurities becomes better.

The reagent for measuring reticulocytes and platelets preferably further contains a polyvalent anion for suppressing nonspecific staining of erythrocytes. Examples of the polyvalent anion include sulfate ion, phosphate ion, carbonate ion, and polyvalent carboxylate ion. Examples of compounds that can supply these ions include citric acid, sulfuric acid, phosphoric acid, EDTA, and alkali metal salts thereof. The reagent of this invention can contain these 1 type, or 2 or more types as a polyvalent anion.
The polyvalent anion is preferably contained in the reagent so that the proportion of the total anion component in the reagent of the present invention is 50% or more, preferably 70% or more.
By containing the above-mentioned polyvalent anion, nonspecific staining of erythrocytes can be suppressed, and discrimination between reticulocytes and platelets and erythrocytes can be facilitated.

  The reagent for measuring reticulocytes and platelets can contain a buffer for keeping the pH constant. The buffer may be contained at a concentration of about several mM to 100 mM. Although it will not specifically limit if it is normally used as a buffering agent, For example, carboxylates, phosphate, a good buffer, taurine, a triethanolamine etc. can be used according to desired pH.

  The reticulocyte and platelet measuring reagent preferably have a pH of 6.0 to 11.0, more preferably 7.0 to 10.0, and even more preferably 8.0 to 9.5. If the pH is too lower than the above range, red blood cells are weakened and easily hemolyzed, and the amount of disrupted red blood cells that are contaminants may increase. If the pH is too higher than the above range, acidic functional groups on the erythrocyte membrane may be dissociated and easily bind to a cationic dye, and nonspecific staining of erythrocytes or disrupted erythrocytes may increase. In addition, when the compound which can supply said polyvalent anion has buffer capacity, such a compound can also be used as a buffering agent.

The reagent for measuring reticulocytes and platelets preferably has an osmotic pressure of 150 to 600 mOsm / kg, more preferably 200 to 300 mOsm / kg. If the osmotic pressure is within such a range, the hypotonic hemolysis of erythrocytes can be prevented because it is closer to the physiological osmotic pressure.
In order to maintain the above osmotic pressure, the reticulocyte and platelet measuring reagent of the present invention can contain an osmotic pressure compensating agent. As the osmotic pressure compensator, those usually used can be used, and examples thereof include alkali metal salts such as propionic acid, and saccharides such as glucose and mannose. In addition, if the ratio of the total anion component in the reagent is less than 50%, an alkali metal halide or alkaline earth metal halide such as NaCl can be used. The osmotic pressure compensating agent can be used alone or in combination of two or more. In addition, when the osmotic pressure of the reagent can be adjusted to the above range using the polyvalent anion and the buffer, the osmotic pressure compensating agent may not be used.

  The reagent for measuring reticulocytes and platelets can contain a staining accelerator for promoting the permeability of the dye to cells. Examples of the dyeing accelerator include surfactants, and cationic surfactants are particularly preferable. Preferred cationic surfactants have the following formula (II):

(Wherein R ₁₁ is an alkyl group having 8 to 12 carbon atoms; R ₁₂ , R ₁₃ and R ₁₄ are the same or different and are an alkyl group having 1 to 6 carbon atoms; Y ⁻ is an anion)
It is represented by
In the above formula (II), the alkyl group having 8 to 12 carbon atoms may be either linear or branched, such as octyl, decyl, lauryl, cetyl, myristyl, etc. Can be mentioned.
Examples of the alkyl group having 1 to 6 carbon atoms are the same as those described for the above formula (I).
As the anion of Y ⁻ , bromine ion or chlorine ion is preferable.

  Preferred cationic surfactants of formula (II) above are decyltrimethylammonium bromide (DTAB), octyltrimethylammonium bromide (OTAB), lauryltrimethylammonium chloride (LTAC), cetyltrimethylammonium chloride (CTAC), myristyltrimethylammonium bromide (MTAB). These cationic surfactants may be used alone or in combination of two or more.

  The above cationic surfactant can promote the staining of reticulocytes and platelets when the reticulocyte and platelet measurement reagent is mixed with the sample, and the reagent of the present invention at a concentration that does not cause red blood cell hemolysis. It is preferable to contain in. Such concentration varies depending on the type of the cationic surfactant, but is usually about 50 to 20000 ppm. For example, when DTAB is used, the concentration in the reagent of the present invention is preferably about 500 to 3000 ppm. For example, when LTAC is used, the concentration in the reagent is preferably about 100 to 500 ppm.

  The reagent for reticulocyte and platelet measurement may contain a preservative such as 2-pyridylthio-1-oxide sodium and β-phenethyl alcohol, in addition to the above components.

The reagent for measuring reticulocytes and platelets includes the above-mentioned first dye and second dye, and optional components such as polyvalent anion, buffer, osmotic pressure compensator, staining accelerator, preservative and the like in the appropriate solvent. It can be produced by dissolving at an appropriate concentration. The solvent is not particularly limited as long as these components can be dissolved stably, and water, water-soluble organic solvents and the like can be mentioned. As the water-soluble organic solvent, for example, ethanol, dimethyl sulfoxide, ethylene glycol, or a mixed solution thereof can be used.
It does not specifically limit as an order which melt | dissolves said each component in a solvent, It can be made to melt | dissolve in arbitrary orders. In addition, each of the above components can be separately dissolved in an appropriate solvent in advance, and the respective solutions can be mixed before use. Such forms are also within the scope of the present invention. For example, when the first dye and the second dye are unstable in an aqueous solution, the dye is dissolved in a water-soluble organic solvent and mixed with an aqueous solution containing other components at the time of use. Can do. In addition, the first dye and the second dye can be dissolved in the same water-soluble organic solvent, or can be dissolved in another water-soluble organic solvent.

  The reagent for measuring reticulocytes and platelets produced in this way can be mixed with the sample and reacted to prepare a measurement sample, thereby staining reticulocytes and platelets that may be contained in the sample. . In this specification, the specimen refers to blood (whole blood, etc.) collected from living organisms, particularly mammals including humans, bone marrow fluid, and samples obtained by diluting them with an appropriate solution such as a buffer solution. Means.

  The mixing ratio (volume ratio) between the reticulocyte and platelet measuring reagent and the sample is preferably reagent: sample = 100: 1 to 1000: 1. The reaction temperature between the reagent and the sample is preferably about 25 to 50 ° C., more preferably about 35 to 45 ° C., and the reaction time varies depending on the type of the dye, but is preferably about 10 seconds to 5 minutes. More preferably, it is about 20 seconds to 2 minutes, and more preferably about 20 seconds to 60 seconds.

Next, the measurement sample prepared as described above is irradiated with light, and the scattered light and fluorescence emitted from the cells in the measurement sample are measured. As an apparatus for irradiating light, a flow cytometer is preferable. When a flow cytometer is used, the measurement sample is introduced into the flow cell of the flow cytometer, and light is irradiated to the cells in the measurement sample flowing in the flow cell.
The light source of the flow cytometer to be used is not particularly limited, and a light source having a wavelength suitable for excitation of the first and second dyes (for example, near 600 to 680 nm) can be used. Examples of the light source include a red semiconductor laser and a He—Ne laser. In particular, a semiconductor laser is very inexpensive as compared with a gas laser and is preferable.

  Scattered light emitted from the cells by irradiating light in the flow cytometer as described above may be either forward scattered light (light receiving angle of 0 to 20 °) or side scattered light (light receiving angle of 90 °). . Further, the forward scattered light may be either forward low-angle scattered light (light receiving angle of 1 to 5 °) or forward high-angle scattered light (light receiving angle of 6 to 20 °). It is known that scattered light is a parameter that reflects information related to cell size.

  Regarding the fluorescence emitted from the cells by irradiating light with the flow cytometer as described above, an appropriate light receiving wavelength can be selected according to the dye to be used.

  By creating a scattergram based on the scattered light and fluorescence obtained as described above, reticulocytes and platelets are distinguished from other blood cell components and contaminants such as fat particles. Can be detected. Reticulocytes and platelets detected in this way can also be counted. Appropriate analysis software is preferably used for detection and / or counting of reticulocytes and platelets.

In another aspect of the present invention, at least one platelet selected from the group consisting of a first reagent containing a first dye for staining the reticulocytes, and capri blue, nile blue, and brilliant cresyl blue. A reagent kit for measuring reticulocytes and platelets is provided, which comprises a second reagent containing a second dye for staining the blood. The kit can further contain a third reagent containing a buffer for keeping the pH during measurement constant.
The same buffer as described above can be used.
These first reagent, second reagent, and third reagent may be obtained by dissolving the first dye, the second dye, and the buffering agent in an appropriate solvent.

In still another aspect of the present invention, a first reagent containing a buffer for keeping the pH constant during measurement, a first dye for staining reticulocytes, capri blue, Nile blue, and brilliant cresyl blue And a reagent kit for measuring reticulocytes and platelets comprising a second reagent containing a second dye for staining at least one platelet selected from the group consisting of:
The same buffer as described above can be used.
These 1st reagent and 2nd reagent may melt | dissolve said buffer agent, and 1st pigment | dye and 2nd pigment | dye in the appropriate solvent, respectively.

  The above-mentioned reagent kit for reticulocyte and platelet measurement may further contain a polyvalent anion, an osmotic pressure compensator, a staining accelerator, a preservative and the like which can be optionally contained in the measurement reagent of the present invention. Good. These components may be contained in the first reagent, the second reagent, or the third reagent described above, or may be contained in the kit as other reagent components.

A sample for measurement can be prepared by mixing each reagent of the reticulocyte and platelet measurement reagent kit with the sample. The order of mixing each reagent of the measurement reagent kit of the present invention and the sample is not particularly limited. The mixing ratio (volume ratio) of these reagents is preferably the sum of each reagent in the reagent kit: analyte = 100: 1 to 1000: 1.
The same applies to the method of preparing a measurement sample, measuring scattered light and fluorescence, and detecting reticulocytes and platelets based on them.

The present invention further includes a first dye for staining reticulocytes, and a second dye for staining platelets that are at least one selected from the group consisting of capri blue, Nile blue, and brilliant cresyl blue. , Prepare a sample for measurement by mixing the sample,
Irradiate the cells in the obtained measurement sample with light, measure the scattered light and fluorescence emitted from the cells,
It is also a reticulocyte and platelet measuring method including detecting reticulocytes and platelets based on detected scattered light and fluorescence.

The present invention will be described in more detail according to the following examples, which are not intended to limit the scope of the present invention.
Reference Examples 1-15
Here, reagents were prepared using various dyes, and the platelet staining ability of each dye was confirmed. The composition of the reagent is as follows.
(1) Dye for staining dye solution platelets Concentration ethylene glycol 1 L as described below
(2) Diluent tricine (buffer) 1.8 g
Trisodium citrate dihydrate (polyvalent anion) 29g
Lauryltrimethylammonium chloride (LTAC) 0.15 g
1L of purified water
(Adjusted to pH 9.0, osmotic pressure 200 mOsm / Kg · H ₂ O)

As dyes for staining platelets, the following dyes were used at the following concentrations. In parentheses, the final concentration when the reagent and the sample are mixed is shown. The chemical formulas of these dyes are as shown in FIG.
Reference Example 1 Capri Blue GON (manufactured by Chroma) 20.5 ppm (0.4 ppm)
2 Nile blue chloride (manufactured by Tokyo Chemical Industry Co., Ltd.) 20.5ppm (0.4ppm)
3 Brilliant cresyl blue ALD (manufactured by Sigma) 51.25 ppm (1 ppm)
4 Azure A (manufactured by Sigma) 102.5ppm (2ppm)
5 Azure D (Sigma) 102.5ppm (2ppm)
6 Azure C (Sigma) 102.5ppm (2ppm)
7 Methylene blue NNX (Sigma) 102.5ppm (2ppm)
8 Cresyl violet acetate (manufactured by Sigma) 1025ppm (20ppm)
9 Basic Green 5 (Tokyo Chemical Industry Co., Ltd.) 1025ppm (20ppm)
10 Methylene blue (Sigma) 102.5ppm (2ppm)
11 New methylene blue (manufactured by Croma) 1025ppm (20ppm)
12 Toluidine Blue (manufactured by Tokyo Chemical Industry) 102.5ppm (2ppm)
13 Oxazine 750 (Nacalai) 102.5 ppm (2 ppm)
14 Oxazine 1 (manufactured by Exciton) 1025 ppm (20 ppm)
15 Oxazine 4 (manufactured by Exciton) 1025 ppm (20 ppm)

1 mL of the obtained diluted solution was dispensed into a tube and heated in a 40 ° C. water bath.
To this was added 20 μL of the obtained dye solution and 5 μL of healthy human whole blood as a specimen, reacted at 40 ° C. for 25 seconds, led to the detection part of a flow cytometer having an excitation light source of 633 nm, and in the measurement sample The cells were irradiated with excitation light, scattered light signals and fluorescence signals emitted from the cells were detected, and the signals obtained were analyzed to measure platelets in the measurement sample.

The scattergrams obtained by these measurements are shown in FIGS. 1-1, 1-2, and 1-3. The scattergram 1 is obtained by taking the forward scattered light intensity on the vertical axis and the fluorescence intensity on the horizontal axis, and the scattergram 2 is obtained by converting the vertical axis of the scattergram 1 into log. A region where platelets appear in scattergram 2 is shown by a solid line.
From the results shown in FIG. 1, when Capri blue, Nile blue or Brilliant cresyl blue is used as the second dye for staining platelets, the fluorescence intensity of platelets is higher than when other dyes are used. It can be seen that the blood cell component can be distinguished better. In addition, when contaminants such as lipid particles are mixed in the blood, a population of contaminants appears at a low fluorescence intensity position on the scattergram, so Capri Blue and Nile are used as dyes for staining platelets. It can be seen that by using blue or brilliant cresyl blue, it is possible to distinguish between platelets and contaminants.

Example 1
Here, a reticulocyte and a platelet measuring reagent were prepared using a first dye for staining reticulocytes and a second dye for staining platelets, and reticulocytes and platelets were measured. The composition of the reagent is as follows.
(1) Dye for staining reticulocytes in dye solution Second dye for staining platelets with concentrations described below Concentration as described below (2) Tricine (buffer) 1.8 g
Trisodium citrate dihydrate (polyvalent anion) 29g
Lauryltrimethylammonium chloride (LTAC) 0.15 g
1L of purified water
(Adjusted to pH 9.0, osmotic pressure 200 mOsm / Kg · H ₂ O)

  As a first dye for staining the above reticulocytes, a dye represented by the following formula was used. The first dye was contained in the dye solution so that the final concentration when the reagent and the sample were mixed was 0.5 ppm, 6 ppm, or 10 ppm.

  Capri blue GON, Nile blue chloride, and brilliant cresyl blue ALD were used as the second dye for staining the platelets. The second dye was contained in the dye solution so that the final concentrations when the reagent and the sample were mixed were capri blue GON 1 ppm, nile blue chloride 0.5 ppm, and brilliant cresyl blue ALD 1.5 ppm, respectively. . In addition, healthy human whole blood, crushed red blood cell-containing blood, and lipid-containing blood were used as specimens. The measurement was performed in the same manner as in Reference Examples 1-15.

The obtained scattergrams are shown in FIGS. 2-1, 2-2 and 2-3. Scattergram 1 is obtained by taking the forward scattered light intensity on the vertical axis and the fluorescence intensity on the horizontal axis, scattergram 2 is obtained by converting the vertical axis of scattergram 1 into log, and scattergram 3 is represented by scattergram 2 This is an enlarged view of the area around the platelet appearance area. A region where reticulocytes appear in scattergram 1 is indicated by a solid line, and a region where platelets appear in scattergram 3 is indicated by a solid line.
For comparison, scattergrams in the case where the second dye for staining platelets is not added to the dye solution are also shown in FIGS. 2-1, 2-2, and 2-3.

  From the results shown in FIG. 2, the concentration of the first dye for staining reticulocytes is stained in the range of 0.5 to 10 ppm, compared with the case where the second dye for staining platelets is not added to the reagent. Secondly, it can be seen that platelets and reticulocytes can be better distinguished by the inclusion of a pigment, even if disrupted erythrocytes or lipids are present.

Example 2
Here, in the reticulocyte and platelet measurement reagent used in Example 1, the first dye for staining the reticulocyte is a final concentration of 0.5 ppm, 6 ppm, or 10 ppm when the reagent and the sample are mixed. The second dye for staining platelets is contained in the dye solution so that the final concentration when the reagent and the sample are mixed is capri blue GON 0.5 ppm, 1 ppm or 2 ppm. I let you. The sample used was the same as in Example 1, and the measurement was performed in the same manner as in Example 1.

  The obtained scattergrams are shown in FIGS. 3-1, 3-2 and 3-3. Scattergram 1 is obtained by taking the forward scattered light intensity on the vertical axis and the fluorescence intensity on the horizontal axis, scattergram 2 is obtained by converting the vertical axis of scattergram 1 into log, and scattergram 3 is represented by scattergram 2 This is an enlarged view of the area around the platelet appearance area. A region where reticulocytes appear in scattergram 1 is indicated by a solid line, and a region where platelets appear in scattergram 3 is indicated by a solid line. From this result, when the 1st pigment | dye for dye | staining the reticulocyte used in Example 1 is 0.5 ppm-10 ppm and capri blue GON is 0.5-2.0 ppm, lipid and crushing as a contaminant It can be seen that even in the presence of red blood cells, reticulocytes and platelets can be more clearly distinguished from contaminants.

Example 3
Here, in the reticulocyte and platelet measurement reagent used in Example 1, the first dye for staining the reticulocyte is a final concentration of 0.5 ppm, 6 ppm, or 10 ppm when the reagent and the sample are mixed. So that the final concentration when the reagent and the sample are mixed is 0.1 ppm, 0.5 ppm, or 2 ppm, when the reagent and the sample are mixed. Contained. The sample used was the same as in Example 1, and the measurement was performed in the same manner as in Example 1.

  The obtained scattergrams are shown in FIGS. 4-1, 4-2 and 4-3. Scattergram 1 is obtained by taking the forward scattered light intensity on the vertical axis and the fluorescence intensity on the horizontal axis, scattergram 2 is obtained by converting the vertical axis of scattergram 1 into log, and scattergram 3 is represented by scattergram 2 This is an enlarged view of the area around the platelet appearance area. A region where reticulocytes appear in scattergram 1 is indicated by a solid line, and a region where platelets appear in scattergram 3 is indicated by a solid line. From this result, when the 1st pigment | dye for dyeing the reticulocyte used in Example 1 is 0.5 ppm-10 ppm and Nile blue chloride is 0.5-2.0 ppm, as a contaminant, lipid and crushing It can be seen that even in the presence of red blood cells, reticulocytes and platelets can be more clearly distinguished from contaminants.

Example 4
Here, in the reticulocyte and platelet measurement reagent used in Example 1, the first dye for staining the reticulocyte is a final concentration of 0.5 ppm, 6 ppm, or 10 ppm when the reagent and the sample are mixed. So that the final concentration when the reagent and the specimen are mixed is brilliant cresyl blue ALD 1 ppm, 1.5 ppm or 2 ppm. Contained. The sample used was the same as in Example 1, and the measurement was performed in the same manner as in Example 1.

  The obtained scattergrams are shown in FIGS. 5-1, 5-2 and 5-3. In these tables, “BCB” indicates brilliant cresyl blue ALD. Scattergram 1 is obtained by taking the forward scattered light intensity on the vertical axis and the fluorescence intensity on the horizontal axis, scattergram 2 is obtained by converting the vertical axis of scattergram 1 into log, and scattergram 3 is represented by scattergram 2 This is an enlarged view of the area around the platelet appearance area. A region where reticulocytes appear in scattergram 1 is indicated by a solid line, and a region where platelets appear in scattergram 3 is indicated by a solid line. From this result, when the first dye for staining the reticulocytes used in Example 1 is 0.5 ppm to 10 ppm and brilliant cresyl blue ALD is 1.0 to 2.0 ppm, lipids as contaminants It can be seen that reticulocytes and platelets can be more clearly distinguished from contaminants even in the presence of ruptured erythrocytes.

The scattergram when staining human blood with reagents containing various oxazine dyes is shown. The scattergram when staining human blood with reagents containing various oxazine dyes is shown. The scattergram when staining human blood with reagents containing various oxazine dyes is shown. The scattergram when dye | staining human blood using the reticulocyte of this invention which changed the density | concentration of the 1st pigment | dye for dyeing reticulocytes variously, and the reagent for platelet measurement is shown. The scattergram when dye | staining human blood using the reticulocyte of this invention which changed the density | concentration of the 1st pigment | dye for dyeing reticulocytes variously, and the reagent for platelet measurement is shown. The scattergram when dye | staining human blood using the reticulocyte of this invention which changed the density | concentration of the 1st pigment | dye for dyeing reticulocytes variously, and the reagent for platelet measurement is shown. Using the reagent for measuring reticulocytes and platelets of the present invention in which the concentration of the first dye for staining reticulocytes and the concentration of capri blue as the second dye for staining platelets are variously changed, human blood The scattergram when dyed is shown. Using the reagent for measuring reticulocytes and platelets of the present invention in which the concentration of the first dye for staining reticulocytes and the concentration of capri blue as the second dye for staining platelets are variously changed, human blood The scattergram when dyed is shown. Using the reagent for measuring reticulocytes and platelets of the present invention in which the concentration of the first dye for staining reticulocytes and the concentration of capri blue as the second dye for staining platelets are variously changed, human blood The scattergram when dyed is shown. Human blood using the reticulocyte and platelet measuring reagent of the present invention in which the concentration of the first dye for staining reticulocytes and the concentration of Nile blue as the second dye for staining platelets are variously changed. The scattergram when dyed is shown. Human blood using the reticulocyte and platelet measuring reagent of the present invention in which the concentration of the first dye for staining reticulocytes and the concentration of Nile blue as the second dye for staining platelets are variously changed. The scattergram when dyed is shown. Human blood using the reticulocyte and platelet measuring reagent of the present invention in which the concentration of the first dye for staining reticulocytes and the concentration of Nile blue as the second dye for staining platelets are variously changed. The scattergram when dyed is shown. Using the reticulocyte and platelet measuring reagent of the present invention in which the concentration of the first dye for staining reticulocytes and the concentration of brilliant cresyl blue as the second dye for staining platelets are variously changed, The scattergram when blood is stained is shown. Using the reticulocyte and platelet measuring reagent of the present invention in which the concentration of the first dye for staining reticulocytes and the concentration of brilliant cresyl blue as the second dye for staining platelets are variously changed, The scattergram when blood is stained is shown. Using the reticulocyte and platelet measuring reagent of the present invention in which the concentration of the first dye for staining reticulocytes and the concentration of brilliant cresyl blue as the second dye for staining platelets are variously changed, The scattergram when blood is stained is shown.

Claims (16)

  A first dye for staining reticulocytes and a second dye for staining platelets, wherein the second dye is selected from the group consisting of capri blue, Nile blue and brilliant cresyl blue A reagent for measuring reticulocytes and platelets, which is at least one dye.   The reagent for measuring reticulocytes and platelets according to claim 1, wherein the first dye is a nucleic acid-staining cyanine dye. The first dye has the following formula:

Wherein R ₁ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or —CH ₂ (CHR ₅ ) _x OR ₆ ;
R ₂ and R ₃ are the same or different and are each a hydrogen atom, a halogen atom, a cyano group, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, an aryl group, or an aralkyl group;
R ₄ is an alkyl group having 1 to 6 carbon atoms, —CH ₂ (CHR ₇ ) _y OR ₈ , an aryl group or an aralkyl group;
R ₅ and R ₇ are each a hydrogen atom or a hydroxyalkyl group having 1 to 3 carbon atoms;
R ₆ and R ₈ are each a hydrogen atom, an acyl group, or an alkyl group having 1 to 3 carbon atoms;
Z is a sulfur atom, an oxygen atom, a selenium atom or CR ₉ R ₁₀ ;
R ₉ and R ₁₀ are the same or different from each other and each is an alkyl group having 1 to 3 carbon atoms;
n is an integer of 1 or 2;
x and y are each an integer of 0 to 3;
X ⁻ is an anion)
The reagent for measuring reticulocytes and platelets according to claim 2 represented by   The reticulocyte and platelet measuring reagent according to any one of claims 1 to 3, further comprising a polyvalent anion for suppressing non-specific staining of erythrocytes.   The reticulocyte and platelet measuring reagent according to any one of claims 1 to 4, having a pH of 6.0 to 11.0.   The reticulocyte and platelet measuring reagent according to any one of claims 1 to 5, wherein the osmotic pressure is 150 to 600 mOsm / kg.   The reagent for measuring reticulocytes and platelets according to any one of claims 1 to 6, comprising a staining accelerator for promoting the permeability of the dye.   The reagent for measuring reticulocytes and platelets according to claim 7, wherein the staining accelerator is a cationic surfactant.   The reticulocyte and platelet measuring reagent according to any one of claims 1 to 8, wherein the second dye stains platelets in a distinguishable manner from lipid particles.   The reagent for reticulocyte and platelet measurement according to any one of claims 1 to 8, wherein the second dye stains platelets in a distinguishable manner from crushed red blood cells.   A first reagent containing a first dye for staining reticulocytes and a second reagent containing a second dye for staining platelets, the second dye being capri blue, nile blue and brilliant cre A reagent kit for measuring reticulocytes and platelets, which is at least one dye selected from the group consisting of Sil Blue.   The reagent kit for reticulocyte and platelet measurement according to claim 11, further comprising a third reagent containing a buffer for keeping the pH constant during measurement.   A first reagent containing a buffer for keeping the pH constant during measurement, and a second reagent containing a first dye for staining reticulocytes and a second dye for staining platelets, A reagent kit for reticulocyte and platelet measurement, wherein the second dye is at least one dye selected from the group consisting of capri blue, nile blue and brilliant cresyl blue. A specimen is mixed with a first dye for staining reticulocytes, a second dye for staining at least one platelet selected from the group consisting of capri blue, Nile blue, and brilliant cresyl blue. Prepare a sample for measurement,
Irradiate the cells in the obtained measurement sample with light, measure the scattered light and fluorescence emitted from the cells,
A method for measuring reticulocytes and platelets, comprising detecting reticulocytes and platelets based on the detected scattered light and fluorescence.   The method for measuring reticulocytes and platelets according to claim 14, wherein the measurement sample is introduced into a flow cell of a flow cytometer, and light is irradiated to cells in the measurement sample flowing in the flow cell.   The method according to claim 14 or 15, wherein the detected reticulocytes and platelets are further counted.

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