JPH0413969A - Reagent for measuring leucocyte and hemoglobin in blood - Google Patents

Abstract

PURPOSE:To enable the maintaining of hemoglobin stbly for a long time without containing cayanogen by incorporating a cation-based surfactant selected from a group comprising a composition of a specified quaternary ammonium salt or a specified pyridinium salt. CONSTITUTION:A cation based surfactant is contained as selected from a group comprising a composition of a quaternary ammonium salt as given by the formula I or of a pyridinium salt as given by the formula II at a concentration that enables the eluting of red corpuscle in blood to oxidize hemoglobin. In the formulas. R1 represents an alkyl, alkenyl or alkynyl group of 8-20C. R2-R4 represent a alkyl, alkenyl or alkynyl group of 1-8C. X<-> represents an anion group and n represents an integer of 7-19. This enables the maintaining of hemoglobin stably for a long time without containing cyanogen.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a reagent for measuring leukocytes and hemoglobin in a blood sample.

[Prior Art and Problems to be Solved by the Invention] Measuring the number of white blood cells and hemoglobin in a blood sample is extremely important for clinical diagnosis of white oil disease, anemia, and the like.

First, the basic method for determining the number of white blood cells is the multiplication method using microscopy and observation. However, in visual arithmetic, Tur
After hemolyzing the red blood cells with K solution and staining them for white blood cells, the white blood cells must be checked one by one on a computer board and examined, which requires a lot of effort and time.

By the way, in determining the number of white blood cells in a blood sample, a method using an automatic blood analyzer is also widely used. Normally, the white blood cell count is determined by an automatic blood analyzer.
First, a red blood cell lysing agent containing surfactant etc. is applied to the blood sample.
By adding l'ffi, red blood cells in the blood sample are selectively lysed to prepare a sample for white blood cell measurement in which only white blood cells remain. This is done by passing through a narrow passage or pore provided in the white blood cell and detecting an electrical or optical signal generated by a detection unit corresponding to each individual white blood cell.

Furthermore, in recent years, by improving red blood cell lysing agents, the deformation of leukocytes is minimized, and the difference in signal intensity obtained allows leukocytes to be separated into granulocytes and monocytes.

Devices for classifying and counting lymphocytes and other classes have also been realized. By using this automatic blood analyzer #L, it is possible to classify and quantify leukocytes much more easily than the above-mentioned visual counting method.

On the other hand, hemoglobin is usually measured using the cyanmethemoglobin method. In the cyanmethemoglobin method, a red blood cell lysing agent such as a nonionic surfactant is added to a blood sample, and the hemoglobin contained in the red blood cells is eluted and the spun hemoglobin is oxidized by the action of an oxidizing agent such as potassium cifericyanide. Finally, cyanide ions are added to convert methemoglobin to cyanmethemoglobin (HiCN).
) A temperature-stable sample for hemoglobin measurement is prepared. Hemoglobin sensitivity is measured by a method of measuring the absorbance of the prepared hemoglobin measurement sample at a specific wavelength. This method has been hailed as an international standard method. The sample prepared by this method is a very stable substance, and since hemoglobin is oxidized using an oxidizing agent, it takes more than ten minutes to complete the oxidation. For this reason, in automatic blood analyzers, the time required for oxidation is shortened by improving the composition of the red blood cell hemolytic agent, and the reaction is completed in several tens of seconds, making it possible to measure hemoglobin at 10°C. Furthermore, it is also possible to measure white blood cells at the same time. However, since the cyanmethemoglobin method uses cyanide, which is a poisonous substance, handling of the reagent is dangerous, and the waste liquid after measurement must be disposed of after decomposing the cyanide using a substance such as primary sodium hypochlorite. There is a lot of floor space, and waste liquid disposal is a hassle.

(lO) Therefore, red blood cells are lysed using only a nonionic surfactant, oxyhemoglobin (HbO2) is converted to Kk without oxidizing hemoglobin to methemoglobin, and #! f Method for measuring absorbance at constant wavelength (oxyhemoglobin method)
will also be held. r, sea urchins are turning into ℃. Since the oxyhemoglobin method does not use cyanide, there is no danger in handling reagents, and there is no trouble in waste liquid treatment.

However, the 2-oxyhemoglobin method has the problem that blood samples with a high methemoglobin content cannot be measured correctly. This is because methemoglobin is not converted to oxyhemoglobin.

As an example, Table 1 shows comparative data of measurement results between the oxyhemoglobin method and Example 1 of the present invention when the methemoglobin content of the blood sample was changed.Methemoglobin gold' increased in the oxyhemoglobin method. The hemoglobin level decreases accordingly.

As a method to solve this problem, Otani et al.
(Anionic surfactant) and Triton X-100 (
Hemoglobin sinuses containing nonionic surfactants are used in clinical biochemistry (C1
in, BiOChem, ) Volume 15, 83 (1982
) is taught at ℃. In this method, red blood cells are lysed by the action of SLS and Triton x-ioo, and the eluted hemoglobin is converted to methemoglobin by the action of SLS.
Sometimes converted to SLS hemoglobin. In this method, methemoglobin in the blood without being affected by goji σ)
It is possible to aim for hemoglobin of 9 degrees, and since it does not contain cyanide, there is no need for troublesome waste liquid treatment. However, with this method, it is not possible to starve leukocytes at the same time as hemoglobin measurement. In addition, 1 Japanese Patent Publication No. 1986-148
569 r cyanide-free hemoglobin reagent", hemoglobin k 1
) Is there a reagent that can be used to determine lij, or can this reagent be used to measure hemoglobin and white blood cells at the same time? It can only be measured.

As described above, with the conventional technology, it is not possible to determine white blood cells at the same time as hemoglobin measurement using a reagent that does not contain cyanide, and furthermore, it is not possible to accurately determine hemoglobin in a blood sample with a high methemoglobin content (σ). There wasn't.

Table 1. Hemoglobin (Ill Mlj constant fruit (oxyhemoglobin method VS Example 1) Methemoglobin content Oxyhemoglobin method Example 10% 16.6t/l 13.6
&/A50% 11.65'/l 1
3.1'/1100% 9.7 t/l
13.6 W-/, the present inventor has previously filed a patent application as Japanese Patent Application No. 1-275280 for a reagent for solving these problems.

This is more important than strictly specifying the type and varus of cationic surfactants, nonionic surfactants, and oxidizing agents used as red blood cell lysing agents. It is a reagent that sometimes performs hemoglobin σ) elution, metefaction, and hemoglobin concentration (5) and also simultaneously measures white blood cells. However, since this reagent does not contain a component that stabilizes methemoglobin, it is difficult to prepare a measurement sample as shown in Comparative Example 4 in Table 2. ! ! There is a possibility that the hemoglobin value changes slightly over time.

This is due to the fact that in a fully automatic type of sample preparation and a device that automatically performs definition, the time from sample θ to definition is short, and since it is specified as a discrete sound, it is not done in the order of intervals, which is an advantage of the previous invention. Although it is denied, it is not σノ.

On the other hand, in the current market, in order to lower the cost of 1 mud foot device), test TF = +? 'l-made? Semi-automatic devices are also used, in which the device only performs measurements by humans.

In a semi-automatic type device, the time spent in the measurement chamber after sample preparation is irregular, and there is usually a variation of several minutes to several tens of minutes from sample preparation to ff1li determination.

For use in these devices, it is necessary to stabilize the hemoglobin value for at least several tens of minutes immediately after sample preparation.

As described above, in the conventional technology, leukocytes are determined at the same time as hemoglobin using a reagent that does not contain cyanide, and furthermore, hemoglobin in a blood sample can be accurately measured by measuring the methemoglobin content, and hemoglobin can be measured stably over a long period of time. It was not possible to obtain a sample for use.

[Means and effects for solving the problem] In order to solve the problems in the above-mentioned prior art, the present inventor has made intensive research and has created the following reagent.

That is, the following reagent for conditionally excreted white blood cells in ml and for determining hemoglobin level is characterized by not containing cyanide and keeping hemoglobin stable for a long period of time. It is a reagent.

1) Hemolyzes red blood cells in the blood and dilutes hemoglobin.
At least one cationic surfactant selected from the group consisting of pyridinium salt (imitation b) compositions.

Structure a R1:08-C2o alkyl group, alkenyl group, or alkynyl group.

R2-R4: CelC8 alkyl group, alkenyl group, or alkynyl group.

X: anionic group structure b n: 7-190) Number to 1.

X: Anionic group 11] Refers to at least one type selected from the group consisting of cationic, nonionic, and amphoteric surfactants having the structure c-ey.

(15) Structure C R□: 08-C2o alkyl group, alkenyl group, or alkynyl group.

R2-R3: C□-C8el C8 alkyl group nyl group.

Or alkynyl group ~C alkyl group R2~F13: C□~C8el C8 alkyl group nyl group.

or an alkynyl group 11i) Contains at least one hemoglobin stabilizer selected from the group consisting of the hemoglobin stabilizers shown in structure t'), and its degree is 11 degrees as shown in structure t').

t″] Imidazole and its derivatives 1m degree 0.001
~20ff/l R□, R2 nee H or 10□~c4 lower alkyl group 1-f3 nee H or -G1~c4 lower alkyl group or phenyl group iV) Solution where l ranges from 3.0 to 9o To be.

■) The quaternary ammonium salt & concentration is 0.1 to 15.
Must be within the range of 0g/L.

Vl) The total degree of pyridinium salt is 0.1 to 15
．． Must be within the 0 (58) P/1ty) range.

vll) The total resistance of the nonionic surfactant is 0.1 to
Must be within the range of 15.05'/l.

vi+D The total concentration of surfactants in both the above notes is 0.1 to 1.
5.0f/lσ) range.

IX) The total concentration of the cation interface agent is 0.1
~15.0? /4σ] range.

By appropriately combining the components in the above reagents, white blood cells can be fractionated into two or three types, such as lymphocytes; monocytes, eosinophils, basophils, and neutrophils. .

As mentioned above, the hemoglobin in the red blood cells is eluted by the action of a suitable red blood cell lysing agent, and the heme iron in the bathed hemoglobin is removed by the action of the red blood cell lysing agent and the oxygen in the solution. The methemoglobin is oxidized by the action of a suitable oxidizing agent, and the resulting methemoglobin is stabilized by the action of a conventional methemoglobin stabilizing agent such as cyanide ions, and the absorbance of the stabilized hemoglobin sample is determined. It is done by doing. However, the presence of this conventional methemoglobin stabilizer does not include the cyanomethemoglobin stabilizer (methemoglobin is mt + the cyanmethemoglobin mentioned above), which is also the cause of the waste liquid treatment problems mentioned above.

Compared to oxyhemoglobin, it is indeterminate over time,
In addition, it has not been used to determine 1Ulj of °C hemoglobin because the absorbance changes depending on the pH of the solution. The present inventors previously created a reagent in Japanese Patent Application No. 1-275280 for stabilizing methemoglobin, which contains the above-mentioned methemoglobin stabilizer. With this reagent, mainly due to the action of the quaternary ammonium salt or pyridinium salt, the amount of particles in the ffl+ liquid sample is increased. Selectively lyses and elutes hemoglobin in red blood cells.

Most of the released hemoglobin undergoes denaturation (change in steric structure) during hooking, and the heme iron (0) in the oxygen hemoglobin dissolved in the reagent changes from 21 to trivalent to acid methemoglobin.

When a single type of cationic surfactant is used,
Although there is a similar complex to methemoglobin made by the method described above,5 this reagent also has the following properties.

Cationic, nonionic, amphoteric surfactants, etc.? By adding an appropriate amount, the degree of denaturation of hemoglobin can be adjusted and stable methemoglobin can be obtained. Further p) II7) effects are resolved by adding buffers as described below. The mechanism of action that stabilizes methemoglobin is not clear, but it is likely that the single molecule structure σ
By allowing different numbers of interfacial agents to act on hemoglobin.

It is thought that one effect is to fix the degree of denaturation (change in steric structure) to methemoglobin at a certain level. In the reagent disclosed in Japanese Patent Application No. 1-275280, leukocytes remain without being destroyed, so that the number of white blood cells σ) can be determined as oJ.

In the reagent of the present invention, the above-mentioned hemoglobin stabilizer is added to further stabilize methemoglobin.

What is the effect of hemoglobin stabilizers?

It is shown in Table 2.

The effect FM- of the hemoglobin stabilizer of the present invention is not clear, but it is probably in the molecular structure of the hemoglobin stabilizer. The lone pair of electrons in the chamber atom chelates to the heme iron in methemoglobin.

It is thought that as a result, methemoglobin is stabilized.

With this reagent, it is possible to fractionate into 2 or 6 leukocytes by specifying the surfactant resistance more precisely.

The composition 91 of the reagent of the present invention is shown below.

Composition Example 1 9 degree range lauryltrimethylammonium 0.1-15.05' chloride (quaternary ammonium salt) lauryldimethylaminoacetic acid beta 0.1-15.
0? Yin (Ampholytic surfactant) 4-phenylimidazole O, CI 01~
2. Of phosphoric acid & impact
1/15~1/60M Sodium chloride 1.0~10
．． 0? purified water
Composition Example 2 Concentration range of myristyl trimethyl ammonia 0.1 to 12. O
ff Mubromide (Quaternary ammonium salt) Polyoxyethylene nonylphenyl 0.1-12.
05' ether (nonionic interfacial additive) 4-phenylimidazole o, ooi~2. Oz Phosphate buffer 1/15 to 1/60M Sodium chloride 1.0 to 10.0 fi' Purified water 1 to composition example 6 Sensitivity range 4-7 Enylimidazole o, ooi to 2. Oz Phosphate buffer 1715~1/60M Sodium chloride 1.0~10.0g Purified water 1 Composition examples 1~6 are quaternary ammonium salts, surfactants such as amphoteric surfactants, and 03 action. The hemoglobin that was eluted by dissolving the red blood cells was converted to meth and stabilized, and further stabilized for a long time by the action of 4-phenylimidazole.

In Composition Example 1, the combination of a quaternary ammonium salt and an amphoteric surfactant makes it possible to measure leukocytes. In Composition Example 2, the combination of a quaternary ammonium salt and a nonionic surfactant makes it possible to measure leukocytes at least It is designed so that it can be fractionated into two parts.

In Composition Example 6, leukocytes can be fractionated into at least six types by combining two types of quaternary ammonium salts.

The total power of the quaternary ammonium salt listed in 60 in the claims is 0,61 to 12 in the case of 1 composition example B, which is the sum of 1 part of lauryltrimethylammonium chloride and 1 part of cetyltrimethylammonium chloride. ．．
01') G-degree bend σ no Koto means.

Composition Example 4 - Degree Range Uryl Trimeter Ammonium 1. o~10.0g-Muchloride imidazole 0.3~20.0? Phosphate buffer 1715~1/60M Sodium chloride Purified water 1.0~10. Oz 1 is used to methelate and stabilize hemoglobin with an amphoteric surfactant, and further stabilized hemoglobin for a long time by the action of a hemoglobin stabilizer.

It is also possible to fractionate -1 blood cells into two or more fractions by adjusting the quaternary ammonium salt and each surfactant. Composition Examples 1 to 4 Smell "C, IJ"
The stimulant is used to adjust the pH of the bath solution to any pH within the range of 3.0 to 90, and any other suitable buffer such as citric acid, maleic powder, Tris, etc. It is not limited to phosphoric acid, and any buffering agent may be used. Furthermore, the preferred pH range is 5.0 to 8.0. If the pH is below 6.0, the damage to white blood cells will increase and it will be difficult to determine white blood cells (Mi+).
If it becomes 90 or more, the stability of hemoglobin over time deteriorates. IJium is used to adjust the electrical conductivity to a level that can be measured by an automatic blood analyzer, and it can be any inorganic or organic salt that dissociates into ions in a water bath. .

The concentration of each component indicated in the claims is such that it can be determined by simply adding blood to the reagent σ), unless other diluents are required. There are, but
When used in a conventional device that mixes a diluent and a lysis reagent at a fixed ratio to determine hemoglobin and white blood cells, each surfactant and other components should be adjusted according to the mixing ratio of the device. It is also possible to change the sensitivity. In that case, each component indicated in the claims refers to the concentration in the mixed solution of the diluent and the lysis reagent.

According to the present invention, 1] Hemoglobin and white blood cells can be measured in one sample] u
11) Blood samples containing methemoglobin can also be measured.

(2S111) Since it does not contain cyanide, there is no need for troublesome waste liquid treatment.

1) A reagent with stable hemoglobin for a long time can be supplied.

Furthermore, when this reagent is used in an automatic blood analyzer.

Since it is no longer necessary to have a separate sample preparation section for hemoglobin measurement and a sample preparation section for regular use, the apparatus is simple and the cost is low.

[Actual hf reli]

A reagent having the following composition was prepared as 1iJi8I, and Table 2 shows the effect of 1--when a hemoglobin stabilizer was added.

Lauryltritylammonium 1.25P chloride lauryldimethylaminoacetic acid 1.005'
Tine purified water t The hemoglobin change sound and change rate in Table 2 were calculated using the following formula.

Hemoglobin change amount 2 Hemoglobin measurement value 60 minutes after sample preparation - After cultivation 2
Hemoglobin measurement value at 0 seconds Hemoglobin measurement value 2 (Hemoglobin change amount/Hemoglobin measurement value at 20 seconds of sample preparation)
I used it.

Comparative Example 1 in Table 2 was measured using the cyanmethemoglobin method (international standard method), and as it takes time to oxidize hemoglobin, the hemoglobin value was 20 seconds after sample preparation as shown in Table 2. There is a big change between 60 minutes and 60 minutes. Comparative Example 2 is used in an automatic blood analyzer.

This is an automated cyanmethemoglobin method, and the change in size is sufficiently small.

Comparative Example 6 shows an example of hemoglobin measurement when methemoglobin was prepared using a single surfactant (lauryltrimethylammonium chloride) except for lauryl dimethylamine acetate betaine in the above composition σ.Methemoglobin was stabilized. Because of this, the hemoglobin value changes significantly between 20 seconds and 60 minutes (σ) after sample preparation.

In addition to the composition of Comparative Example 6, betaine lauryldimethylaminoacetate, which is an amphoteric surfactant, is added. In Comparative Example 4 (composition example of Japanese Patent Application No. 1-275280), the amount of change is smaller than in Comparative Example 6.

Furthermore, the hemoglobin stabilizer fK which is a reagent of the present invention:
In the added compositions (Examples 1-4), the variable lid was compared to Comparison 11.
This is further reduced compared to P114.

FIG. 1 shows a correlation diagram between the results of measuring the number of white blood cells using the conventional method CF-800C (manufactured by Tsukaa Medical Electronics Co., Ltd.) and the results of measuring the number of white blood cells using the reagent of Example 1.

Correlation coefficient γ=1.000. Regression line V=1.001X+
It shows a very high correlation with 0.331°C.

Figure 8PL2 shows a contrast diagram between the hemoglobin value measurement results using the conventional method [cyanmethemoglobin method (international standard method)] and the hemoglobin value measurement results using the reagent of Example 1 (30).

Correlation coefficient γ=0.998. Regression straight #y=1.013x+
C1,095 shows a very unusual rIA.

As shown in Table 1, due to an increase in methemoglobin,
Even when the hemoglobin measurement value by the oxyhemoglobin method decreases, the hemoglobin measurement value using the reagent of Example 1 does not change and shows a constant value.

Example 5 A reagent with the following composition was prepared.

once lauryl trityl ammonium i, soy
Chloride cetyl tritylammonium chloride 0.4110
Ride phosphate buffer 1/25M
(pH 6.0) Adjusted to around 13 fA) 4-phenylimidazole 100η (3s Purified water 1) Example 6 A reagent with the following composition was prepared.

m degree lauryl trityl ammonium 3.20? Chloride cetyl tritylammonium chloride 0.20? Loride Phosphate Slow #Elephant 1/25M
(pH 6,0) Around 16! ! 4-Phenylimidazole 1[]OIn
g Purified water 16th 6th
Figures 1 to 5 show the leukocyte particle size distribution when using the reagents of Examples 1, 5, and 6, respectively. In Figures 6 to 5, the vertical axis represents the relative frequency of blood cells, and the horizontal axis represents the relative intensity (
It represents the size of blood cells. Example 10 When using reagents.

As shown in FIG. 6, leukocytes exhibit a unimodal particle size distribution. When the reagent of Example 5 is used, the leukocytes exhibit a particle size distribution divided into two regions, region A and region B, as shown in FIG. Region A is a population of lymphocytes, and region B is a population of white blood cells other than lymphocytes. When the reagent of Example 6 was used, the leukocytes showed a particle size distribution divided into six regions: region A, region A, and region A, as shown in FIG. Region A is a population of lymphocytes, region C is a population of monocytes, eosinophils, and basophils, and region A is a population of neutrophils.

〔Effect of the invention〕

As described above, according to the reagent of the present invention, not only can the measured value be the same as that of the conventional method, but also 1) hemoglobin and leukocytes can be measured in one sample, and leukocytes can also be fractionated.

11] Blood samples with high methemoglobin content can also be measured correctly.

111] Since it does not contain cyanide poison, there is no need for waste liquid treatment.

IV) A reagent that keeps hemoglobin stable for a long time can be supplied (3
3.

[Brief explanation of drawings]

FIG. 1 is a correlation diagram between the white blood cell!!2 measurement results using the conventional method CF-800 (manufactured by Toa Medical Electronics Co., Ltd., 'Cm11 constant) and the white blood cell count measurement results using the reagent of Example 1. The figure is a correlation diagram between the hemoglobin 1111 determination result by the conventional method [cyanmethemoglobin method (international standard method)] and the hemoglobin value determination result by the reagent of Example 1. 17) is a graph showing leukocyte particle size distribution when using the reagent of Example 1.5.6. In Figures 3 to 5, the vertical axis represents the relative strength of blood cells, and the horizontal axis represents the relative intensity of the blood cell signal (i.e., the size of blood cells) obtained when blood cells are measured by an automatic blood analyzer. . In FIG. 6, white blood cells have a unimodal particle size distribution. The white blood cells shown in FIG. 4 show a particle size distribution divided into two regions, region A and region B. Region A is a population of lymphocytes, and region B is a population of white blood cells other than lymphocytes. Region A is a population of lymphocytes, region C is a population of monocytes, eosinophils, and basophils, and region A is a population of neutrophils. Patent applicant Toa Medical Electronics Co., Ltd.

Claims (1)

[Claims] 1. A reagent for counting leukocytes in blood and measuring hemoglobin concentration, which satisfies the following conditions, and is characterized by containing cyanide and keeping hemoglobin stable for a long time. reagent. i) A cationic surfactant selected from the group consisting of compositions of quaternary ammonium salts (structure a) or pyridinium salts (structure b) at a concentration capable of hemolyzing red blood cells in blood and oxidizing hemoglobin. Contain at least one type of. Structure a ▲There are mathematical formulas, chemical formulas, tables, etc.▼ R_1: C_8 to C_2_0 alkyl group, alkenyl group, or alkynyl group, R_2 to R_4: C_1 to C_8 alkyl group, alkenyl group, or alkynyl group, X^-: Anion Group structure b ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ n: An integer from 7 to 19, X^-: Anionic group ii) From the group consisting of cationic, nonionic, and amphoteric surfactants having structures c to e Contain at least one selected type. Structure c ▲There are mathematical formulas, chemical formulas, tables, etc.▼ R_1: C_8 to C_2_0 alkyl group, alkenyl group, or alkynyl group, R_2 to R_3: C_1 to C_8 alkyl group, alkenyl group, or alkynyl group, X^-: Anion Base structure d R_1-R_2-(CH_2CH_2O)_n
-HR_1: C_8 to C_2_0 alkyl group, alkenyl group, or alkynyl group, R_2: O, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, or COO, n: Integer structure of 10 to 50 e ▲ Numerical formulas, chemical formulas, tables, etc. ▼ R_1: C_8 to C_2_0 alkyl group, R_2 to R_3: C_1 to C_8 alkyl group, alkenyl group, or alkynyl group iii) At least one type of hemoglobin stabilizer selected from the group consisting of structure f) and its concentration is as shown in f). f) Imidazole and its derivatives, concentration 0.001-20g/l ▲Mathematical formulas, chemical formulas, tables, etc. are available▼ R_1, R_2; -H or lower alkyl group of -C_1 to C_4 R_3: -H or -C_1 to C_4 Lower alkyl group or phenyl group iv) The solution should have a pH range of 3.0 to 9.0. v) The total concentration of the quaternary ammonium salt is 0.1 to 15.
Must be within the range of 0g/l. vi) The total concentration of the pyridinium salt is 0.1 to 15.0.
Must be within the g/l range. vii) The total concentration of the nonionic surfactant is from 0.1 to
Must be within the range of 15.0g/l. viii) The total concentration of the amphoteric surfactant is 0.1 to 15
．． Must be within the range of 0g/l. ix) The total concentration of the cationic surfactant is 0.1 to 1
Must be within the range of 5.0g/l. 2. Contains at least two types selected from the group consisting of quaternary ammonium salts, and the total concentration of quaternary ammonium salts is 0.1 to 12.0 g/
2. The reagent according to claim 1, which is in the range of 1 and capable of fractionating leukocytes into at least two types. 6. Contains at least one type selected from the group consisting of quaternary ammonium salts, the total concentration of quaternary ammonium salts is in the range of 0.1 to 12.0 g/l, and consists of a nonionic surfactant. Contains at least one type selected from the group and has a total concentration of nonionic surfactants of 0.1 to 12.0
The reagent according to claim 1, which is in the range of g/l and is capable of fractionating leukocytes into at least two groups. 4. A group consisting of amphoteric surfactants, including at least one type selected from the group consisting of quaternary ammonium salts, with a total concentration of quaternary ammonium salts in the range of 0.1 to 12.0 g/l; Contains at least one type selected from
2. The reagent of claim 1, wherein the total concentration of amphoteric surfactant is in the range 0.1-12.0 g/l and is capable of fractionating leukocytes into at least two groups. 5. Contains at least one type selected from the group consisting of quaternary ammonium salts, the total concentration of quaternary ammonium salts is in the range of 0.1 to 12.0 g/l, and consists of a cationic surfactant. A cationic surfactant containing at least one type selected from the group, having a total concentration of cationic surfactants in the range of 0.1 to 5.0 g/l, and capable of fractionating leukocytes into at least two types. 1 reagent. 6. Contains at least one type selected from the group consisting of quaternary ammonium salts, with a total concentration of quaternary ammonium salts in the range of 0.1 to 12.0 g/l, and from the group consisting of pyridinium salts. containing at least one second class selected from
2. A reagent according to claim 1, wherein the total concentration of pyridinium salts is in the range 0.6-10.0 g/l and is capable of fractionating leukocytes into at least two groups. 7. Contains at least two types selected from the group consisting of quaternary ammonium salts, the total concentration of quaternary ammonium salts is in the range of 2.2 to 5.0 g/l, and leukocytes are divided into six types. 2. The reagent of claim 1. 8. Contains at least one type selected from the group consisting of quaternary ammonium salts, the total concentration of quaternary ammonium salts is in the range of 6.0 to 12.0 g/l, and consists of a nonionic surfactant. at least one type selected from the group, and the total concentration of nonionic surfactants is 1.0 to 12.
The reagent according to claim 1, which has a concentration in the range of 0 g/l and is capable of dividing leukocytes into three types. 9. A group consisting of amphoteric surfactants, containing at least one type selected from the group consisting of quaternary ammonium salts, with a total concentration of quaternary ammonium salts in the range of 1.0 to 10.0 g/l; Contains at least one type selected from
2. The reagent of claim 1, wherein the total concentration of amphoteric surfactants is in the range 0.1 to 10.0 g/l and is capable of fractionating leukocytes into three groups. 10. Contains at least one type selected from the group consisting of quaternary ammonium salts, with a total concentration of quaternary ammonium salts in the range of 1.0 to 10.0 g/l, and selected from the group consisting of pyridinium salts. The reagent according to claim 1, which contains at least one kind selected from the following, has a total concentration of pyridinium salts in the range of 1.0 to 5.0 g/l, and is capable of fractionating leukocytes into three types.