JP4806342B2 - Reagent and reagent kit for immature leukocyte analysis - Google Patents

Description

  The present invention relates to a reagent and a reagent kit for classifying and counting white blood cells contained in a sample collected from a living body.

Blood cells are made in the bone marrow, differentiate and mature from immature cells, and migrate to peripheral blood. In healthy people, young leukocytes (young leukocytes) do not appear in the peripheral blood, but immature leukocytes appear in the peripheral blood of patients with leukemia, bone marrow metastasis of cancer, severe infections, etc. There are things to do. For this reason, measuring mature leukocytes and immature leukocytes in a biological sample is extremely important in diagnosing the above-mentioned diseases.

As a reagent for measuring leukocytes, a reagent disclosed in Patent Document 1 is known. A measurement sample that is a mixture of this reagent and a biological sample is introduced into a flow cytometer, and the mature and immature leukocytes in the specimen are classified based on the optical information obtained by irradiating light of a specific wavelength. , Each can be counted. It is also possible to subdivide immature leukocytes into myeloblasts and immature granulocytes and to count them. However, when a sample containing immature leukocytes is processed using this reagent, damage to immature leukocytes such as myeloblasts may progress depending on the treatment conditions, and the classification accuracy may decrease. Therefore, in order to accurately classify and count immature leukocytes in a sample using this reagent, it is necessary to strictly manage processing conditions such as reaction temperature and reaction time.

Japanese Patent Laid-Open No. 10-206423

It is an object of the present invention to accurately analyze immature leukocytes and mature leukocytes such as myeloblasts and immature granulocytes, and to provide a reagent for immature leukocyte analysis that has a wider allowable range of sample processing conditions than before. And providing a reagent kit.

The present invention relates to immature leukocytes comprising a surfactant that damages cell membranes of erythrocytes and mature leukocytes in peripheral blood , a solubilizer that contracts damaged blood cells, a sugar, and a dye that stains nucleic acids. Analytical reagents are provided.

The present invention also includes a surfactant that damages cell membranes of erythrocytes and mature leukocytes, a solubilizer that contracts damaged blood cells, and an osmotic pressure regulator, and has an osmotic pressure of 150 to 600 mOsm / kg. A reagent for analyzing immature leukocytes for analyzing immature leukocytes contained in peripheral blood, comprising a first reagent having an electric conductivity of less than 6 mS / cm and a second reagent containing a dye for staining nucleic acid Provide kit.

The present invention also includes a surfactant that damages cell membranes of erythrocytes and mature leukocytes, a solubilizing agent that contracts damaged blood cells, a first reagent containing sugar, and a dye that stains nucleic acid. Provided is a reagent kit for analyzing immature leukocytes for analyzing immature leukocytes contained in peripheral blood, including a second reagent.

According to the present invention, immature leukocytes and mature leukocytes can be analyzed with high accuracy, and a reagent and reagent kit for immature leukocyte analysis with a wider allowable range of sample processing conditions than before are provided.

When the reagent for analyzing immature leukocytes (hereinafter also simply referred to as a reagent) of this embodiment is used, the leukocytes contained in the sample can be classified into mature leukocytes and immature leukocytes, and each can be counted. Moreover, mature leukocytes can be subdivided into lymphocytes, monocytes and granulocytes, and each can be counted. In particular, when this reagent is used, immature leukocytes can be subdivided into immature granulocytes and myeloblasts, and each can be accurately counted.

In the present specification, immature leukocytes refer to immature leukocytes present in bone marrow but not in peripheral blood in healthy individuals. For example, it refers to myeloblast, promyelocyte, myelocyte, posterior myelocyte and the like. Promyelocytes, myelocytes and posterior myelocytes may be immature granulocytes. The myeloblast also includes leukocyte hematopoietic progenitor cells such as myeloid stem cells (CFU-GEMN), neutrophil / macrophage colony forming cells (CFU-GM), and eosinophil colony forming cells (CFU-EOS). .

Samples that will be subjected to the measurement is peripheral blood.

The reagent of the present embodiment includes a surfactant that damages cell membranes of erythrocytes and mature leukocytes, a solubilizing agent that contracts damaged blood cells, and a nucleic acid staining dye. When the biological sample and the reagent are mixed, the cell membrane of blood cells contained in the sample is damaged by the action of the surfactant. This surfactant damages the cell membranes of erythrocytes and mature leukocytes, but does not substantially damage the cell membranes of immature leukocytes. Damaged blood cells such as red blood cells and mature white blood cells contract by the action of solubilizers. Since the cell membrane of immature leukocytes is hardly damaged, cell contraction by solubilizing agents is less likely to occur than erythrocytes and mature leukocytes. Damaged blood cell nuclei are stained by the action of the pigment, but immature leukocytes are hardly stained.

As the surfactant, for example, a polyoxyethylene nonionic surfactant can be used. Specifically, those having the following structural formula can be used.

In particular, it is preferable to use polyoxyethylene (16) oleyl ether, polyoxyethylene (20) lauryl ether, polyoxyethylene (15) oleyl ether, or the like.

Although the preferable density | concentration of surfactant contained in a reagent changes with kinds of surfactant, for example, when using polyoxyethylene (16) oleyl ether, 1000-50000 ppm is preferable and 10000-35000 ppm is more preferable. Surfactants may be used alone or in combination of two or more surfactants.

As the solubilizer, for example, a sarcosine derivative or a salt thereof, a cholic acid derivative, methyl glucanamide, or the like can be used.

The sarcosine derivative has the following structural formula.

The cholic acid derivative has the following structural formula.

Methyl glucanamide has the following structural formula.

When a sarcosine derivative or a salt thereof is used, the concentration in the reagent is preferably 200 to 3000 ppm. When a cholic acid derivative is used, it is preferably 100 to 10,000 ppm. When methyl glucanamide is used, it is preferably 1000 to 8000 ppm.

Specific examples of the sarcosine derivative or a salt thereof include sodium N-lauroyl sarcosinate, sodium lauroylmethyl β-alanine, lauroyl sarcosine and the like. Specific examples of cholic acid derivatives or salts thereof include CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate), CHAPSO ([(3-cholamidopropyl) dimethylammonio ] -2-hydroxy-1-propanesulfonate) and the like. Specific examples of methyl glucanamide include MEGA8 (octanoyl-N-methylglucamide), MEGA9 (nonanoyl-N-methylglucamide), MEGA10 (decanoyl-N-methylglucamide) and the like.

In addition to these, n-octyl β-glucoside, sucrose monocaprate, N-formylmethylleucylalanine, and the like can be used as a solubilizer. When using these, it is preferable that the density | concentration in a reagent is 10-50000 ppm. A solubilizer may be used independently and may use two or more sorts of solubilizers together.

The dye is not particularly limited as long as it specifically stains a nucleic acid, but is preferably a fluorescent dye. By using such a dye, red blood cells having no nucleus are hardly stained, and white blood cells having a nucleus are strongly stained. Red blood cells and white blood cells can be discriminated based on the difference in staining intensity. In addition, mature leukocytes having damaged cell membranes that can penetrate the dye are strongly stained by the dye, but immature leukocytes are hardly stained. Based on this difference in staining intensity, mature leukocytes and immature leukocytes among the leukocytes can be discriminated. The kind of the dye is appropriately selected depending on the light to be irradiated. For example, when a helium-neon laser or a red semiconductor laser is used as a light source, it is preferable to use a dye having the following structural formula.

（式中、R1Iは水素原子または低級アルキル基を示し；R2I及びR3Iは、それぞれ同一または異なって、水素原子、低級アルキル基または低級アルコキシ基を示し；R4Iは水素原子、アシル基または低級アルキル基を示し；R5Iは水素原子または置換されてもよい低級アルキル基を示し；Zは硫黄原子、酸素原子、または低級アルキル基で置換された炭素原子を示し；nIは１または２を示し；XI-はアニオンを示す。）

(Wherein R1I represents a hydrogen atom or a lower alkyl group; R2I and R3I are the same or different and each represents a hydrogen atom, a lower alkyl group or a lower alkoxy group; R4I represents a hydrogen atom, an acyl group or a lower alkyl group; R5I represents a hydrogen atom or an optionally substituted lower alkyl group; Z represents a sulfur atom, an oxygen atom, or a carbon atom substituted with a lower alkyl group; nI represents 1 or 2; XI- Represents an anion.)

In the formula, the lower alkyl group for R1I is a linear or branched alkyl group having 1 to 6 carbon atoms. Examples thereof include a methyl group, an ethyl group, a propyl group, a butyl group, an isobutyl group, a sec-butyl group, a ter-butyl group, a pentyl group, and a hexyl group. Among these, a methyl group and an ethyl group are preferable.
The lower alkyl group in R2I and R3I is the same as described above, and the lower alkoxy group means an alkoxy having 1 to 6 carbon atoms. For example, a methoxy group, an ethoxy group, a propoxy group and the like can be mentioned. R2I and R3I are preferably hydrogen atoms.
The acyl group in R4I is preferably an acyl group derived from an aliphatic carboxylic acid. Specifically, an acetyl group, a propionyl group, etc. are mentioned, Among these, an acetyl group is preferable. The lower alkyl group is the same as described above.
The lower alkyl group in R5I is the same as described above, and the optionally substituted lower alkyl group may be substituted with 1 to 3 hydroxyl groups, halogen atoms (fluorine, chlorine, bromine or iodine) or the like. Means a good lower alkyl group. Of these, a methyl group and an ethyl group substituted with one hydroxyl group are preferred.
The lower alkyl group for Z is the same as described above, and Z is preferably a sulfur atom.
Anions in X- are halogen ions (fluorine, chlorine, bromine or iodine ions), boron halide ions (BF4-, BCl4-, BBr4-, etc.), phosphide compound ions, halogen oxygenate ions, fluorosulfate ions, methyl Examples thereof include a sulfate ion, an aromatic ring halogen or a tetraphenylboron compound ion having an alkyl group having a halogen as a substituent. Of these, bromine ion or BF4- is preferred.

As specific examples of the dye (I), the following dyes are preferable.
Dye A

  Dye B

  Dye C

In addition to the above dyes, propidium iodide, ethidium bromide, ethidium-acridine heterodimer, ethidium diazide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, TOTO-1, TO-PRO-1, TOTO- 3, TO-PRO-3, Iodine Green, NK-3975 (Hayashibara Biological Laboratory), NK-1570 (Hayashibara Biological Laboratory), NK-1049 (Hayashibara Biological Laboratory), etc. Preferably used.

The concentration of the dye in the reagent is preferably 0.01 to 500 ppm, more preferably 0.1 to 200 ppm. A pigment | dye may be used independently and may use two or more types of pigment | dyes together.

The osmotic pressure of the reagent is preferably adjusted to 150 to 600 mOsm / kg. In order to adjust the osmotic pressure of the reagent to a desired range, the reagent contains a sugar. By adjusting the osmotic pressure of the reagent using sugar, the myeloblasts are less likely to be damaged even if the reaction time is prolonged or the reaction temperature is increased, and the classification and counting of myeloblasts and mature leukocytes is accurately performed. It can be carried out.

Moreover, sodium chloride can also be contained in a reagent as a substance (osmotic pressure osmotic pressure regulator) which adjusts osmotic pressure. However, if the reagent contains a large amount of sodium chloride, the more the reaction time elapses after the reagent and the sample are mixed, or the higher the reaction temperature, the more the myeloblast damage is promoted. Is dyed with pigment. When myeloblasts are stained, the detected fluorescence intensity becomes equivalent to the fluorescence intensity of mature leukocytes, and the classification accuracy between mature leukocytes and myeloblasts decreases. From the above, the concentration of sodium chloride contained in the reagent is preferably 0.01 to 3 g / L, and more preferably 0 g / L (not included) so as not to adversely affect the measurement.

Although the kind of saccharide | sugar contained in a reagent is not specifically limited, A monosaccharide, polysaccharide, sugar alcohol, etc. can be used. Examples of monosaccharides include glucose and fructose, examples of polysaccharides include arabinose, and examples of sugar alcohols include xylitol, sorbitol, mannitol, and ribitol. The concentration of sugar in the reagent is preferably 10 to 75 g / L, more preferably 20 to 50 g / L. One kind of these sugars may be used, or two or more kinds may be used in combination.

It is preferable to add a buffer to the reagent in order to adjust the pH of the reagent. As the buffer, for example, a Good buffer such as HEPES, a phosphate buffer, and a pH osmotic pressure regulator such as sodium hydroxide can be used. The pH of the reagent is preferably adjusted to 5.0 to 9.0.

Each of the components described above may be housed in the same container, but it is preferable to house them in two or more containers to form a reagent kit. This reagent kit includes a surfactant that damages cell membranes of erythrocytes and mature leukocytes, a solubilizing agent that contracts damaged blood cells and a sugar, and a second reagent that contains a dye. In this case, in order to improve the storage stability of the dye, the dye of the second reagent is preferably dissolved in an organic solvent.

A reagent kit according to another embodiment of the present invention includes a surfactant that damages cell membranes of erythrocytes and mature leukocytes, a solubilizing agent that contracts damaged blood cells, and an osmotic pressure adjusting agent. A reagent and a second reagent containing a nucleic acid staining dye.

The osmotic pressure adjusting agent is added to adjust the osmotic pressure and electrical conductivity of the first reagent to desired values. By using this osmotic pressure adjusting agent, the osmotic pressure of the first reagent is adjusted to 150 to 600 mOsm / kg. The electrical conductivity of the first reagent is adjusted to less than 6 mS / cm, preferably 0.01 to 3 mS / cm, more preferably 0.1 to 2 mS / cm.

As the osmotic pressure adjusting agent, sugar, amino acid or the like can be used. As the amino acid, valine, proline, glycine, alanine and the like can be used, but it is preferable to use either or both of glycine and alanine. As a density | concentration of an amino acid, 1-50 g / L is preferable and 10-30 g / L is more preferable. In addition, when sodium chloride is further added to the reagent as an osmotic pressure adjusting agent, the concentration in the reagent is 0.01 to 3 g / L so as not to affect the myeloblast measurement as described above. Is preferable, and it is more preferable that it is 0 g / L.

The reagent containing each component described above and a biological sample are mixed to prepare a measurement sample, and immature leukocytes can be analyzed using a flow cytometer.

The mixing ratio of the biological sample and the reagent (in the case of a reagent kit, a mixed reagent of all reagents) is preferably 1:10 to 1: 1000. The reaction between the blood cells in the biological sample and the reagent is preferably performed at 20 to 40 ° C. for 3 to 15 seconds. When the reaction temperature is high, the reaction time is shortened, and when the reaction temperature is low, the reaction time is lengthened.

When a flow cytometer is used to measure a sample, light is irradiated onto blood cells in the measurement sample flowing in the flow cell to obtain optical information such as scattered light and fluorescence, and the type of blood cell is specified based on this information. Is done.

Specifically, a flow cytometer as shown in FIG. 1 can be used. Hereinafter, myeloblast measurement will be described in detail based on FIG. 1 as an example of this embodiment.

The measurement sample discharged from the nozzle 6 flows through the orifice portion of the flow cell 23. At this time, blood cells in the sample pass through the orifice part in a line. The light emitted from the light source 21 is applied to the blood cells flowing through the flow cell 23 via the collimating lens 22. By irradiating the blood cells with light, side scattered light, side fluorescence and forward scattered light are generated. Side scattered light is incident on a side scattered light detector (photomultiplier tube) 29 via a condenser lens 27 and a dichroic mirror 28. Side fluorescence enters a side fluorescence detector (photomultiplier tube) 31 through a condenser lens 27, a dichroic mirror 28, a filter 29, and a pinhole plate 30. The forward scattered light is incident on the forward scattered light detector (photodiode) 26 through the condenser lens 24 and the pinhole plate 25.

The forward scattered light signal output from the forward scattered light detector 26, the side scattered light signal output from the side scattered light detector 29, and the side fluorescent signal output from the side fluorescent detector 31 are as follows. Are amplified by the amplifier 32, the amplifier 33, and the amplifier 34, respectively, and input to the analysis unit 35.

The analysis unit 35 calculates the forward scattered light intensity, the side scattered light intensity, and the fluorescence intensity from the received forward scattered light signal, side scattered light signal, and side fluorescent signal. The analysis unit 35 creates a first two-dimensional distribution map with the forward scattered light intensity and the fluorescence intensity as two axes, and displays an area where all white blood cells appear in the sample (total white blood cell area) on the two-dimensional distribution map. Identify. Further, a second two-dimensional distribution map having two axes of the side scattered light intensity and the fluorescence intensity is created for the cells appearing in the whole leukocyte region. On this two-dimensional distribution map, a region where mature leukocytes appear (mature leukocyte region), a region where lymphocytes appear (lymphocyte region), a region where monocytes appear (monocyte region), and a region where granulocytes appear Set (granulocyte region). Furthermore, a region where myeloblasts appear (myeloblast region) and a region where immature granulocytes appear (immature granulocyte region) are specified. The number of cells appearing in the myeloblast region is counted as the number of myeloblasts contained in the sample, and the number of cells appearing in the immature granulocyte region is counted as the number of immature granulocytes contained in the sample. Since myeloblasts have a small cell size and are mononuclear, the forward scattered light intensity is high and the side scattered light intensity is low. In addition, the fluorescence intensity is weak because it is hardly stained as described above. Since immature granulocytes have a large cell size and nuclei are divided, the forward scattered light intensity and the side scattered light intensity are weak. Moreover, since it hardly dyes as mentioned above, the fluorescence intensity is weak.

Example 1
A first reagent A and a second reagent having the following composition were prepared.
<First reagent A>
Polyoxyethylene (16) oleyl ether (Nikko Chemicals) 20000 ppm
Sodium N-lauroyl sarcosinate 500ppm
Arabinose 39.6 g / L
HEPES 10 mM
1L of purified water
The above was mixed, and NaOH was further added to adjust the pH to 7.0. The osmotic pressure of the first reagent A was 280 mOsm / kg, and the electric conductivity was 0.59 mS / cm.
<Second reagent>
Dye A 20ppm
Ethylene glycol 1L

After mixing 980 μL of the first reagent, 20 μL of the second reagent, and 20 μL of blood sample A containing myeloblasts, the mixture was reacted at 33 ° C. for 7 seconds, and then the side scattered light intensity was measured with the flow cytometer shown in FIG. Scattered light intensity and fluorescence intensity were measured. A red semiconductor laser was used as the light source.

A first two-dimensional distribution map with the obtained side scattered light intensity and fluorescence intensity as two axes was created, and the total leukocyte region was identified. This figure is shown in FIG. The number of cells appearing in the total leukocyte region was counted as the total leukocyte count.

For cells appearing in the whole white blood cell region in the first two-dimensional distribution map, create a second two-dimensional distribution map with the side scattered light intensity and fluorescence intensity as two axes, and the side scattered light intensity is small and fluorescent. A region with low intensity was identified as the myeloblast region. This figure is shown in FIG. The number of cells appearing in the myeloblast region was counted as the number of myeloblasts.

In this example, the ratio of myeloblasts to total leukocyte count (myeloblast ratio = myeloblast count / total leukocyte count × 100) was calculated. The myeloblast ratio was 45.8%.

(Example 2)
The myeloblast ratio was calculated in the same manner as in Example 1 except that the reagent and blood sample A were reacted at 35 ° C. The myeloblast ratio was 42.6%. The first two-dimensional distribution diagram created in Example 2 is shown in FIG. 4, and the second two-dimensional distribution diagram is shown in FIG.

(Comparative Example 1)
Polyoxyethylene (16) oleyl ether 24.0 g / L, sodium N-lauroyl sarcosate 1.5 g / L, DL-methionine 20.0 g / L, HEPES 12.0 g / L, 1N-NaOH 0.3 g / L The myeloblast ratio was calculated in the same manner as in Example 1 except that 1 mL of a reagent containing NaCl 4.0 g / L and 3.0 mg / L of dye A and 33 μL of blood sample B were mixed and reacted. The myeloblast ratio was 25.1%. The first two-dimensional distribution diagram created in Comparative Example 1 is shown in FIG. 6, and the second two-dimensional distribution diagram is shown in FIG. The osmotic pressure of this reagent was 350 mOsm / kg, and the electric conductivity was 7.4 mS / cm.

(Comparative Example 2)
The myeloblast ratio was calculated in the same manner as in Comparative Example 1 except that the reagent and blood sample B were reacted at 35 ° C. The myeloblast ratio was 7.2%. The first two-dimensional distribution chart created in Comparative Example 2 is shown in FIG. 8, and the second two-dimensional distribution chart is shown in FIG.

Compared to Comparative Examples 1 and 2, the myeloblasts reacted at 35 ° C. compared to the myeloblast ratio (Comparative Example 1) when reacted at 33 ° C. even though the same blood sample was used. The ratio (Comparative Example 2) showed a very low value. This is because when reacted at 35 ° C., myeloblasts that should appear in the myeloblast region appeared outside the region. That is, when the reagents prepared in Comparative Examples 1 and 2 were used, the higher the reaction temperature, the more stable the myeloblasts were, and the myeloblasts were damaged during the 7-second reaction. Conceivable.

However, from Examples 1 and 2, the myeloblast ratio (Example 1) when reacted at 33 ° C. is a value approximate to the myeloblast ratio (Example 2) when reacted at 35 ° C. Indicated. This shows that even when the reagent prepared in the example and myeloblast were reacted at a high temperature, the myeloblast could be accurately counted without substantial damage. That is, when the reagents prepared in the examples were used, the stability of myeloblasts with respect to temperature was improved and it was difficult to damage. From the above, it was confirmed that myeloblasts in a sample can be accurately measured by using the reagent prepared in the example.

(Example 3)
First reagent B is not arabinose but xylitol 39.56 g / L, polyoxyethylene (16) oleyl ether 25000 ppm instead of 20000 ppm, and N-lauroyl sarcosinate sodium 750 ppm instead of 500 ppm Prepared in the same manner as A. The osmotic pressure of the first reagent B was 280 mOsm / kg, and the electric conductivity was 0.59 mS / cm.

The myeloblast ratio was calculated in the same manner as in Example 1 except that the first reagent B was used instead of the first reagent A, and the blood sample C was used instead of the blood sample A. Example 1 except that the first reagent B is used instead of the first reagent A, the blood sample C is used instead of the blood sample A, and the reagent and the blood sample C are reacted for 12 seconds instead of 7 seconds. The myeloblast ratio was calculated in the same manner as above.

Example 4
The first reagent C was prepared in the same manner as the first reagent B except that 39.52 g / L of arabinose was contained instead of xylitol. The osmotic pressure of the first reagent C was 280 mOsm / kg, and the electric conductivity was 0.62 mS / cm.

The myeloblast ratio was calculated in the same manner as in Example 3 except that the first reagent C was used instead of the first reagent B.

(Example 5)
The first reagent D was prepared in the same manner as the first reagent B except that 23.16 g / L of alanine was contained instead of xylitol. The osmotic pressure of the first reagent D was 280 mOsm / kg, and the electric conductivity was 0.61 mS / cm.

The myeloblast ratio was calculated in the same manner as in Example 3 except that the first reagent D was used instead of the first reagent B.

(Example 6)
The first reagent E was prepared in the same manner as the first reagent B except that 19.52 g / L of glycine was contained instead of xylitol. The osmotic pressure of the first reagent E was 280 mOsm / kg, and the electric conductivity was 0.63 mS / cm.

The myeloblast ratio was calculated in the same manner as in Example 3 except that the first reagent E was used instead of the first reagent B.

(Comparative Example 3)
The myeloblast ratio was calculated in the same manner as in Comparative Example 1 except that blood sample C was used instead of blood sample A. The myeloblast ratio was calculated in the same manner as in Comparative Example 1 except that blood sample D was used instead of blood sample A, and that the reagent and blood sample C were allowed to react for 12 seconds instead of 7 seconds.

The results of Examples 3 to 6 and Comparative Example 3 are shown in FIG. FIG. 10 is a graph showing how much the myeloblast ratio when the sample and the reagent are reacted for 12 seconds is lower than the myeloblast ratio when the sample is reacted for 7 seconds.

From FIG. 10, when the reagent of Comparative Example 3 was used, when the reagent and the sample were reacted for 12 seconds, the ratio of myeloblasts decreased by 20% or more compared to the case of reacting for 7 seconds. This is considered to be because myeloblasts were damaged by the influence of the reagent as the reaction time became longer.

When the reagent prepared in any of Examples 3 to 6 was used, the decrease in myeloblast ratio was suppressed to about 10% compared to the case where the reagent of Comparative Example 3 was used. That is, the stability of myeloblasts was improved by using the reagents of Examples 3 to 6, and myeloblasts were less likely to be damaged in the measurement sample. As described above, it was confirmed that when the reagents of Examples 3 to 6 were used, myeloblasts were hardly damaged and myeloblasts could be measured accurately.

(Example 7)
A first reagent F having the following composition was prepared.
Polyoxyethylene (16) oleyl ether 25000ppm
Sodium N-lauroyl sarcosinate 750ppm
Xylitol 37.0 g / L
HEPES 10 mM
1L of purified water
The above was mixed, and NaOH was further added to adjust the pH to 7.0. The osmotic pressure of the first reagent F was 280 mOsm / kg, and the electric conductivity was 0.64 mS / cm.
The second reagent used was the same as that prepared in Example 1.

The myeloblast ratio was calculated in the same manner as in Example 1 except that the first reagent F was used instead of the first reagent A, and the blood sample D was used instead of the blood sample A. The myeloblast ratio was 45%. The first two-dimensional distribution diagram created in Example 7 is shown in FIG. 11, and the second two-dimensional distribution diagram is shown in FIG. In the second two-dimensional distribution diagram of FIG. 12, a region where mature leukocytes appear (mature leukocyte region) is also set.

The myeloblast ratio of blood sample D was 58.5% when calculated with a microscope. From the fact that the myeloblast ratio measured in Example 7 and the myeloblast ratio calculated with a microscope showed an approximate value, the use of the reagent of Example 7 allowed mature leukocytes and myeloblasts in blood samples to be obtained. It was confirmed that it was possible to accurately discriminate and accurately count myeloblasts.

(Example 8)
The myeloblast ratio was calculated in the same manner as in Example 5 except that blood sample G was used instead of blood sample D. The myeloblast ratio was 5.5%. The first two-dimensional distribution diagram created in Example 8 is shown in FIG. 13, and the second two-dimensional distribution diagram is shown in FIG.

Further, when the myeloblast ratio of blood sample E was calculated with a microscope, the myeloblast ratio was 6.3%. Since the myeloblast ratio measured in Example 8 and the myeloblast ratio calculated by the microscope showed an approximate value, the use of the reagent of Example 8 allowed mature leukocytes and myeloblasts in the blood sample to be obtained. It was confirmed that it was possible to accurately discriminate and accurately count myeloblasts.

Further, a region where immature granulocytes appear in the first two-dimensional distribution map shown in FIG. 13 (immature granulocyte region) is set, and cells appearing in this region are counted as the number of immature granulocytes. . Based on this value, the ratio of immature granulocytes to the total white blood cell count (immature granulocyte ratio) was calculated. The immature granulocyte ratio was 10%.

When the immature granulocyte ratio of blood sample E was calculated with a microscope, the immature granulocyte ratio was 13%. Since the immature granulocyte ratio measured in Example 8 and the immature granulocyte ratio calculated by a microscope showed an approximate value, mature leukocytes and myeloblasts in a blood sample were obtained using the reagent of Example 8. And immature granulocytes were accurately discriminated, and it was confirmed that not only myeloblasts but also immature granulocytes could be accurately counted.

Example 9
The immature granulocyte ratio was calculated in the same manner as in Example 5 except that blood sample F was used instead of blood sample D. The immature granulocyte ratio was 12.4%. The first two-dimensional distribution diagram created in Example 9 is shown in FIG. 15, and the second two-dimensional distribution diagram is shown in FIG.

When the myeloblast ratio and immature granulocyte ratio of blood sample E were calculated with a microscope, the immature granulocyte ratio was 10.8%. Since the immature granulocyte ratio measured in Example 9 and the immature granulocyte ratio calculated by a microscope showed an approximate value, mature leukocytes and immature granules in a blood sample were obtained using the reagent of Example 9. It was confirmed that it was possible to accurately discriminate spheres and accurately count immature granulocytes.

It is a flow cytometer. FIG. 3 is a first two-dimensional distribution diagram in the first embodiment. FIG. 3 is a second two-dimensional distribution diagram in the first embodiment. 6 is a first two-dimensional distribution diagram in Example 2. FIG. 6 is a second two-dimensional distribution diagram in Example 2. FIG. 6 is a first two-dimensional distribution diagram in Comparative Example 1. FIG. 6 is a second two-dimensional distribution diagram in Comparative Example 1. FIG. 6 is a first two-dimensional distribution diagram in Comparative Example 2. FIG. 10 is a second two-dimensional distribution diagram in Comparative Example 2. FIG. It is a graph which shows the result of Examples 3-6 and the comparative example 3. FIG. FIG. 10 is a first two-dimensional distribution diagram in the seventh embodiment. FIG. 10 is a second two-dimensional distribution diagram in the seventh embodiment. 10 is a first two-dimensional distribution diagram in Example 8. FIG. 10 is a second two-dimensional distribution diagram in Example 8. FIG. 10 is a first two-dimensional distribution diagram in Example 9. FIG. 10 is a second two-dimensional distribution diagram in Example 9. FIG.

Explanation of symbols

  6: Nozzle 21: Light source 22: Collimating lens 23: Flow cell 24: Condensing lens 25: Pinhole plate 26: Forward scattered light detector 27: Condensing lens 28: Dichroic mirror 29: Side scattered light detector 30: Pin Hall plate 31: Side fluorescence detector 32: Amplifier 33: Amplifier 34: Amplifier 35: Analysis unit

Claims (15)

A reagent for analyzing immature leukocytes contained in peripheral blood ,
A reagent for analysis of immature leukocytes comprising a surfactant that damages cell membranes of erythrocytes and mature leukocytes, a solubilizing agent that contracts damaged blood cells, a sugar, and a dye that stains nucleic acids. The surfactant has the following structural formula

The reagent according to claim 1, which is a polyoxyethylene nonionic surfactant containing   The reagent according to claim 1 or 2, wherein the solubilizer is at least one selected from the group consisting of a sarcosine derivative, a salt of a sarcosine derivative, a cholic acid derivative, and methyl glucanamide.   The reagent according to any one of claims 1 to 3, wherein the sugar is at least one selected from the group consisting of xylitol, arabinose, glucose, mannitol, sorbitol, and ribitol.   The reagent according to any one of claims 1 to 4, comprising 10 to 75 g / L of the sugar. Sodium chloride containing no reagent according to any one of claims 1 to 5.   The reagent in any one of Claims 1-6 whose pH is 5.0-9.0.   The reagent according to any one of claims 1 to 7, wherein the osmotic pressure is 150 to 600 mOsm / kg. A reagent kit for analyzing immature leukocytes contained in peripheral blood ,
A surfactant that damages the cell membrane of red blood cells and mature white blood cells;
A solubilizing agent that contracts damaged blood cells;
An osmotic pressure adjusting agent,
A reagent kit for immature leukocyte analysis comprising a first reagent having an osmotic pressure of 150 to 600 mOsm / kg and an electric conductivity of less than 6 mS / cm, and a second reagent containing a dye for staining nucleic acid. The reagent kit according to claim 9, wherein the electrical conductivity of the first reagent is 3 mS / cm or less.   The reagent kit according to claim 9 or 10, wherein the osmotic pressure adjusting agent is at least one selected from the group consisting of sugar and amino acid.   The reagent kit according to claim 11, wherein the amino acid is at least one selected from the group consisting of glycine and alanine.   The reagent according to claim 11 or 12, comprising 1 to 50 g / L of the amino acid.   The reagent according to any one of claims 9 to 13, wherein the concentration of sodium chloride in the first reagent is less than 3 g / L. A reagent kit for analyzing immature leukocytes contained in peripheral blood ,
A first reagent containing a surfactant that damages cell membranes of erythrocytes and mature leukocytes, a solubilizing agent that contracts damaged blood cells, and a sugar, and a second reagent that contains a dye that stains nucleic acid Reagent kit for young leukocyte analysis.

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