JP3505621B2 - Leukocyte classification reagent and leukocyte classification method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reagent used for classifying blood cells in the field of clinical examination, and more specifically, it is a method for optically measuring blood cells after lysing red blood cells using a flow cytometer to measure white blood cells. The present invention relates to a reagent used in the method for classifying. It also relates to a white blood cell classification method using the reagent.

[0002]

2. Description of the Related Art Conventionally, as a method for classifying and counting white blood cells, a lysing agent for red blood cells containing a quaternary ammonium salt as a main component among cationic surfactants in a blood diluting solution (hereinafter referred to as hemolytic agent) Was added to dissolve the membranes and cytoplasm of erythrocytes and leukocytes, and the nuclei of remaining leukocytes were measured by the Coulter principle to count the number of leukocytes. This method has a problem that information for classifying the types of white blood cells cannot be obtained because the white blood cells are observed in a contracted state.

In order to solve this problem, the components and the concentration of the hemolytic agent are improved and the reaction of leukocytes is slowed down, so that the difference in the dissolution rate of lymphocytes, monocytes and granulocytes of white blood cells and electrical conduction. There is a method of classifying white blood cells into three according to the degree of difference. However, counting leukocytes in blood by type can contribute to diagnosis of diseases. Therefore, if possible, lymphocytes, monocytes, neutrophils, eosinophils, and basophils can be classified into at least five categories. Recently, there has been a strong demand for a method of classifying into four types excluding basic spheres.

On the other hand, conventionally, a flow cytometer has been used to identify and analyze cells and fine particles due to light scattering. The flow measuring device 4 of this cell measuring device (flow cytometer) shown in FIG. 10 is designed to measure light scattering from sample particles. That is, the sample liquid containing the particles to be measured is guided to the flow measuring device 4, where the laser light from the laser light source 1 is irradiated through the irradiation light focusing lenses 2 and 3, and the forward scattered light is irradiated by the irradiation light stopper. The direct light is stopped therethrough, and only the scattered light passes through the forward scattered light detecting lens 8 and is measured by the forward scattered light detector 9, and the voltage level measured by the detector is input to the analyzer 10. On the other hand, the side scattered light in the flow measuring device 4 is measured by the side scattered light detector 6 through the side scattered light detecting lens 5, and is input to the voltage level analyzer 10 measured by the detector. Based on both voltage levels of the analyzer 10, a two-dimensional distribution chart (scattergram) by the forward and side scattered light is displayed on the display device 11.

However, even when measured by a flow cytometer, as long as the hemolytic agent containing the quaternary ammonium salt type surfactant as the main component is used, the cell membrane of white blood cells is considerably dissolved. The reality is that the number of white blood cells can only be counted and three classifications are difficult.

In the method of classifying white blood cells by measuring forward scattered light and side scattered light using a flow cytometer, a hemolytic agent containing ammonium oxalate as a main component is added to a blood dilution solution to lyse only red blood cells. White blood cells into lymphocytes, monocytes,
It was classified into three types of granulocytes. However, this method requires 20 to 30 minutes to lyse erythrocytes, which is unsuitable as a method for treating a large number of specimens. Moreover, according to this method, the distribution of each leukocyte was not clear, and eosinophils were buried in the distribution of neutrophils, so that the distribution of eosinophils alone could not be separated.

In order to solve the above problems, 3 to 5 leukocytes containing at least one surfactant selected from the group consisting of polyoxyethylene anionic surfactants and polyoxyethylene nonionic surfactants are used. A leukocyte classification reagent for classification has been proposed (see International Patent Application Publication No. W088 / 09504).

However, these polyoxyethylene-based surfactants, both anionic and nonionic, have the following common general formula:

[0009]

[Chemical 1]

That is, the number of moles of ethylene oxide added is relatively large, 8 to 30. This large number of moles of ethylene oxide added reduces the cytolytic power. Also, using this reagent, PF. Also, when a two-dimensional distribution map is obtained from the measurement results by the DC method, the problem remains that lymphocytes, monocytes, and granulocytes are poorly separated, and eosinophils cannot be measured simultaneously.

[0011]

SUMMARY OF THE INVENTION The present invention has been made in view of such conventional problems. When added to a blood diluting solution, white blood cells are formed in spite of lysing red blood cells in a short time. Leukocytes can be directly converted into lymphocytes by measuring the forward scattered light and side scattered light with a flow cytometer after lysing the red blood cells for a certain period of time in the original state or a state close to this that is not damaged by the hemolytic agent. It is an object of the present invention to provide a leukocyte classification reagent (hemolytic agent) that can be classified into four groups, namely, monocytes, eosinophils and neutrophils.

[0012]

The invention according to claim 1 is
Red blood cells were lysed, a leukocyte classification reagent which is characterized in that to enable classify leukocytes by acting on leukocytes, polyoxyethylene (3) alkyl (C ₁₂ -C ₁₃₎ ether sulfate, polyoxyethylene (3) Contains sodium alkyl (C ₁₁ -C ₁₅ ) ether sulfate or polyoxyethylene (3) alkyl (C ₁₁ -C ₁₅ ) ether triethanolamine.

The invention according to claim 2 further comprises a first step of adding the reagent according to claim 1 to the blood sample solution to lyse red blood cells in the blood sample solution to produce hemolyzed blood, and A second step of directing the hemolyzed blood obtained in the first step to a flow cytometer in order to measure light scatter and side light scatter and thereby create a scattergram which classifies white blood cells into a plurality of groups, Is a method of classifying white blood cells.

[0014]

[Function] Anionic surfactants and amphoteric surfactants useful for leukocyte classification reagents, when added to a diluted blood solution,
It lyses erythrocytes in a short period of time, but it is necessary for leukocytes to remain intact or near undamaged.

In order to satisfy the above requirements, the general formula R--O--SO ₂ X (1) (wherein R is an alkyl or alkylene group having 8 to 18 carbon atoms, X is Na, K, NH ₄ or HN (CH ₂
CH ₂ OH) ₃ ) in the alkylsulfate chlorine anion surfactant represented by R has a carbon number of 8 to 18
And the general formula R—O— (CH ₂ CH ₂ O) n—SO ₃ X (2) (wherein R is an alkyl or alkylene group having 8 to 18 carbon atoms, and n is 1 to 5). X is Na, K,
NH ₄ or HN (CH ₂ CH ₂ OH) ₃ ) in the polyoxyethylene anionic surfactant, R has 8 to 18 carbon atoms, and the number of moles of ethylene oxide added is 1 to 5 It is necessary to be.
Hydrogen of an alkyl group or an alkylene group R is greatly involved in cell lysis. Generally, the longer the chain length of R, the stronger the solubilizing power, and the shorter the R chain, the weaker it becomes. However, if the chain length of R becomes long, the solubility in water will decrease, making it difficult to dissolve or suspending hemolyzed blood. The carbon number of R within the above-mentioned limited range is well balanced and satisfies the above requirements. Further, as n increases, hydrophilicity increases, but cell lysing power decreases, and the above-mentioned limited range satisfies the above-mentioned requirements. In addition, X has nothing to do with the reaction to cells. The above description regarding the carbon number of R is represented by the general formula (3)

[0016]

[Chemical 2]

(Wherein R is an alkyl or alkylene group having 8 to 18 carbon atoms, and X is CH ₂ SO ₃ or CO
O and Y is Na, K, NH ₄ or HN (CH ₂ C
H ₂ OH) ₃ ) and the coconut oil fatty acid-based anionic surfactant is also applicable, and the range of 8 to 18 carbon atoms of R satisfies the above requirements. Further, similarly to the above, both X and Y in the general formula (3) are not particularly related to the reaction to cells.

The amphoteric surfactant acts as an anionic surfactant or a cationic surfactant depending on the pH range, but when it is used as a hemolytic agent at a pH of 7 or more, it acts as an anionic surfactant. And general formula (4)

[0019]

[Chemical 3]

In the betaine type amphoteric activator represented by the formula (wherein R is an alkyl or alkylene group having 6 to 21 carbon atoms), the range of 6 to 21 carbon atoms of R satisfies the above requirements. Is.

In the present invention, the blood is diluted with a general diluent for uniformly dispersing the blood before adding the hemolytic agent which is a leukocyte classification reagent. This diluting solution is composed of distilled water in which an appropriate amount of an osmotic pressure adjusting agent, a buffering agent, a chelating agent and a fungicide is dissolved. Diluent Sodium chloride 9.0 g Potassium dihydrogen phosphate 0.91 g Disodium hydrogen phosphate 9.55 g EDTA2Na 0.3 g 1-Hydroxypyridine-2-thione sodium 0.25 g Distilled water 1000 mL

The osmotic pressure adjusting agent has an osmotic pressure at which cells can stably exist, that is, an osmotic pressure of a blood sample solution of 250 to 40.
It is adjusted to 0 mOsm / kgH ₂ O. When the osmotic pressure is higher than this range, the hemolytic power becomes stronger, particularly the erythrocyte hemolysis proceeds, and the neutrophils contract a little. On the other hand, when the osmotic pressure is lower than this range, the hemolytic power is weakened, and especially red blood cell ghost (red blood cell undissolved) increases.

The buffer is preferably a buffer which maintains the pH of the blood sample solution at about 6.8 to 7.6 so that blood cells can stably exist. However, when the blood sample solution is mixed with the lysing agent, the pH must be within a suitable range for use. Therefore, the range may be exceeded if the desired result is not compromised. The anionic surfactant according to the present invention generally has a pH range of 3 to 11, although the suitable use range varies depending on its type, and the amphoteric surfactant acts as a hemolytic agent in the pH range of 7 to 11. Is. When the pH is higher than this range, the hemolytic power becomes strong and the white blood cells are also lysed at the same time as the red blood cells.
On the other hand, if the pH is lower than this range, the hemolytic power will be weakened,
Separation of distribution of each component of white blood cells becomes poor.

The chelating agent is used for forming a chelate with a metal ion in order to prevent the precipitation of the diluent due to the metal ion during long-term storage and use. The antifungal agent is used to suppress the generation of mold and bacteria due to long-term storage of the diluted solution.

Although the amount of the hemolytic agent containing the above-mentioned surfactant as a main component, which is added to the diluted blood solution diluted with the above-mentioned diluent, varies depending on the kind of the surfactant, it is generally a blood sample solution. A low concentration of detergent in it is not enough to lyse all red blood cells and results in more red blood cell ghosts. On the other hand, when the concentration of the surfactant is high, white blood cells are also lysed together with red blood cells. Further, the dissolution time also changes depending on the amount of the hemolytic agent added. When the amount of addition is large, the lysis time is short, but on the other hand, the time during which the white blood cells are stable becomes short. Therefore, it is necessary to properly select the range of the added amount of the surfactant.

[0026]

EXAMPLES The present invention will be described below based on examples. Example 1 Polyoxyethylene (3) alkyl (C ₁₂ which is one of polyoxyethylene-based anionic surfactants as a hemolytic agent
~ C ₁₃ mixture) Sodium ether sulfate (chemical formula C ₁₂ ~
An example of using C ₁₃ -O- and _{(CH 2 CH 2 O) 3} -SO 3 Na). 1.75 g of this anionic surfactant is dissolved in 1000 ml of distilled water to obtain a hemolytic agent (note that sodium chloride may be added to distilled water in an appropriate amount to adjust the osmotic pressure, if necessary). 25 μl of blood was diluted with 1 ml of the diluent having the above-mentioned composition, and 125 μl of the above hemolytic agent
l is added. After 10 seconds, forward and side scattered light intensities were measured with a flow cytometer to obtain a two-dimensional distribution chart (scattergram) shown in FIG. As shown in FIG.
White blood cells were clearly classified into four groups: lymphocytes (1), monocytes (2), neutrophils (3) and eosinophils (4). (0) is the red blood cell ghost. In addition, the above hemolytic agent (concentration 0.175
%) Is the addition amount range to the above-mentioned blood diluting solution 100 to 1
With 000 μl, a two-dimensional distribution map similar to that shown in FIG. 1 was obtained. The object of the present invention can also be achieved by adding the hemolytic agent to the diluent first, or using a solution in which an appropriate amount of the surfactant is directly added to the diluent without diluting it.
Further, with the surfactant of this example, results similar to those shown in FIG. 1 were obtained even in the pH range of 3 to 10. Also regarding the osmotic pressure range, 250 to 400 mOsm / kgH ₂ O
Then, the same result as that shown in FIG. 1 was obtained.

Example 2 Sodium polyoxyethylene (3) alkyl (C ₁₁ -C ₁₅ mixture) ether sulfate, which is one of polyoxyethylene type anionic surfactants, was used as a hemolytic agent, and this anionic surfactant was used. 2.5 g of distilled water 1000
Example 1 except that what was dissolved in ml was used as the hemolytic agent
FIG. 2 shows the result of classifying white blood cells by the same procedure as described above.
As in Example 1, white blood cells were clearly classified into four types. Also,
Addition amount range 50-300 μl (in the case of hemolytic agent with a concentration of 0.25%), pH range 6-10, and osmotic pressure range 250-
With 400 mOsm / kgH ₂ O, similar results to those shown in FIG. 2 were obtained as in the case of Example 1.

Example 3 Number of moles of ethylene oxide added as the active ingredient of hemolytic agents
Polyoxyethylene-based anionic surfactant in the case of 0
Sodium lauryl sulfate (Formula C₁₂H _{twenty five}OSO
₃Na) and 5.0 g of this anionic surfactant
What was dissolved in 1000 ml of distilled water was used as a hemolytic agent
Other than the above, the result of classifying white blood cells by the same procedure as in Example 1
Is shown in FIG. As in Example 1, white blood cells were clearly classified into four types.
Was done. In the case of a hemolytic agent with a concentration of 0.5%, the addition amount range
Range 50-200 μl, pH range 5-8, osmotic pressure range 25
0-400mOsm / kgH₂Even in the case of O in the case of the first embodiment
The same result as that shown in FIG. 3 was obtained.

Example 4 In the polyoxyethylene-based anionic surfactant represented by the general formula (2), the carbon number of R is 8 and 18.
The same leukocyte classification test as in Examples 1 to 3 was carried out for each of n and 3, and both were similar to lymphocytes, monocytes, neutrophils and eosinophils similar to FIGS. 1 to 3. A scattergram in which white blood cells were clearly classified into 4 was obtained.

Comparative Example 1 As in Example 3, using sodium lauryl sulfate as the active ingredient of the hemolytic agent, the effect of pH on the classification of white blood cells was shown in FIG.
And shown in FIG. It can be seen from FIG. 4 that at pH 10, the hemolytic power becomes too strong and the white blood cells and the red blood cells dissolve at the same time, so that only a two-dimensional distribution map that is almost unmeasurable can be obtained. On the other hand, at pH 3, the hemolytic power became too weak and the separation of white blood cells deteriorated, as shown in FIG. 5, and only unsatisfactory results were obtained.

Comparative Example 2 As in Example 2, polyoxyethylene was used as the active ingredient of the hemolytic agent.
Tylene (3) alkyl (C₁₁~ C₁₅Mixture) ether sulfur
Of osmolarity for classification of white blood cells using sodium acidate
About impact 450mOsm / kgH₂O and 220m
Osm / kgH ₂The test was performed at O. Osmotic pressure 45
0 mOsm / kgH₂With O, the hemolytic power becomes too strong,
Red blood cells are hemolyzed, but white blood cells are also hemolyzed.
As the sphere contracts a little, the height shown in FIG.
Only a two-dimensional distribution map that is almost unmeasurable as in the case of pH
I couldn't get it. On the other hand 220 mOsm / kgH₂In O
Hemolysis becomes too weak, especially with many red blood cell ghosts.
Therefore, only unsatisfactory results as shown in Fig. 7 were obtained.
It was Further, the alkyl group R and the number of moles of ethylene oxide n
The same was true when was outside the scope of claims.

Example 5 Palm oil fatty acid methyl taurine sodium (chemical formula R-CON (CH ₃ ) CH ₂ CH ₂ SO ₃ Na), which is one of palm oil fatty acid type anionic surfactants, was used as a hemolytic agent. Here is an example: 5.8 g of this anionic surfactant was dissolved in 1000 ml of distilled water (an appropriate amount of sodium chloride may be added to adjust the osmotic pressure as in Example 1) to obtain a hemolytic agent. The same procedure as in Example 1 was repeated to obtain the two-dimensional distribution chart shown in FIG. As is clear from FIG. 8, leukocytes were clearly classified into four groups: lymphocytes (1), monocytes (2), neutrophils (3) and eosinophils (4). In addition, (0) shows an erythrocyte ghost. Further, as in the case of Example 1, the above-mentioned hemolytic agent has an addition amount range of 50 to 50 at a concentration of 0.58%.
300 μl, pH range 6-9, osmotic pressure range 250-40
With 0 mOsm / kgH ₂ O, similar results to those shown in FIG. 9 were obtained.

Example 6 One of betaine-type amphoteric surfactants as a hemolytic agent,
The following structural formula

[0034]

[Chemical 4]

2-alkyl (C ₈ H ₁₇ -C represented by
₁₈ H ₃₇ mixture) -N-carboxymethyl-N-hydroxyethylimidazolinium betaine is used. 3.6 g of this amphoteric surfactant was dissolved in 1000 ml of distilled water in the same manner as in Example 5 to obtain a hemolytic agent. The same procedure as in Example 5 was repeated to obtain the two-dimensional distribution chart shown in FIG. As is clear from FIG. 9, the white blood cells were clearly classified into 4 as in Examples 1 to 5. Further, as in Example 5, when the concentration of the hemolytic agent was 0.36%, the addition amount range was 75 to 200 μl, the pH range was 7 to 11, and the osmotic pressure range was 250 to 400 mOsm / kgH ₂ O. Results similar to those shown were obtained.

Example 7 With respect to the betaine-type amphoteric surfactant represented by the above general formula (3), the same leukocyte classification test as in Example 6 was carried out for carbon atoms 6 and 21 of the alkyl group of R.
In both cases, a two-dimensional distribution map in which leukocytes were clearly classified into four groups was obtained similar to that shown in FIG.

[0037]

EFFECTS OF THE INVENTION According to the present invention, when added to a blood diluting solution, erythrocytes can be lysed in a short time, and leukocytes can be maintained in their original state where they are not damaged or in a state close thereto, for a certain period of time. With simple steps and configuration,
It can be directly classified into four categories, and can exert a great effect on clinical examination.

[Brief description of drawings]

FIG. 1 is a two-dimensional distribution diagram when polyoxyethylene (3) alkyl (C ₁₂ -C ₁₃ ) sodium ether sulfate is used as a hemolytic agent.

FIG. 2 is a two-dimensional distribution diagram when polyoxyethylene (3) alkyl (C ₁₁ -C ₁₅ ) sodium ether sulfate is used as a hemolytic agent.

FIG. 3 is a two-dimensional distribution diagram when sodium lauryl sulfate is used as a hemolytic agent.

FIG. 4 uses sodium lauryl sulfate as a hemolytic agent,
It is a two-dimensional distribution map when the white blood cells are classified at pH 10.

[FIG. 5] Using sodium lauryl sulfate as a hemolytic agent,
It is a two-dimensional distribution map when white blood cells are classified at pH 3.

FIG. 6 is a two-dimensional distribution chart when white blood cells are classified at an osmotic pressure of 450 mOsm / kgH ₂ O using polyoxyethylene (3) alkyl (C ₁₁ -C ₁₅ ) sodium sulfate as a hemolytic agent.

FIG. 7 is a two-dimensional distribution diagram when white blood cells are classified at an osmotic pressure of 220 mOsm / kgH ₂ O using sodium polyoxyethylene (3) alkyl (C ₁₁ -C ₁₅ ) ether sulfate as a hemolytic agent.

FIG. 8 is a two-dimensional distribution diagram when coconut oil fatty acid methyl taurine sodium is used as a hemolytic agent.

FIG. 9: 2-alkyl (C ₈ -C ₁₈ ) -N as hemolytic agent
FIG. 3 is a two-dimensional distribution diagram when using -carboxymethyl-N-hydroxyethylimidazolinium betaine.

FIG. 10 is an explanatory diagram of an optical system of a flow cytometer using the leukocyte classification reagent of the present invention.

[Explanation of symbols]

0 red blood cell ghost 1 lymphocyte 2 monocytes 3 neutrophils 4 eosinophils

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-3-266999 (JP, A) JP-A-4-13969 (JP, A) JP-A-3-252557 (JP, A) International Publication 88/009504 (WO, A1) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 33/49 G01N 33/48

Claims (2)

(57) [Claims] 1. A reagent for classifying leukocytes, which lyses erythrocytes and acts on leukocytes to classify leukocytes, which comprises polyoxyethylene (3) alkyl (C ₁₂ -C ₁₃ ) ether sulfate. sodium polyoxyethylene (3) alkyl (C ₁₁ -C ₁₅₎ ether sulfate, or polyoxyethylene (3) alkyl (C ₁₁ -C ₁₅₎ reagent containing ether triethanolamine. 2. A first step of adding the reagent of claim 1 to the blood sample solution to lyse red blood cells in the blood sample solution to produce hemolyzed blood; forward light scattering and side light scattering. To produce a scattergram that classifies white blood cells into multiple groups,
A second step of guiding the hemolyzed blood obtained in the first step to a flow cytometer, the white blood cell classification method.