JPH03252557A - Reagent for measuring white cell and hemoglobin in blood - Google Patents

Abstract

PURPOSE:To stabilize hemoglobin over a long period of time without containing cyanide by using a reagent which satisfies specific conditions. CONSTITUTION:The reagent satisfying the conditions, such as inclusion of at least one kind of the cationic surfactants selected from the group consisting of the compsn. of the quaternary ammonium salt of formula 1 (R1 is 8 to 20C alkyl group, alkenyl group, or alkinyl group; R2 to R4 are 1 to 8C alkyl group, alkenyl group or alkinyl group; X<-> is an anion group) or the pyridinium salts of formula 2 ((n) is 7 to 19; X<-> is an anion group) of the concn. to hemolyze, for example, the red cells in blood and to allow the oxidation of the hemoglobin is used in order to calculate the white cells in the blood and to measure the concn. of the hemoglobin.

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 leukemia, anemia, and the like.

The basic method for determining the number of white blood cells is the visual counting method using microscopic observations. However, in visual arithmetic, T'i
After hemolyzing red blood cells with rk solution and staining white blood cells, the white blood cells on the counting board are counted one by one using a microscope, which requires a lot of effort and time.

By the way, when it comes to measuring daily cell counts in blood samples, methods using automatic blood analyzers are also widely used.
To measure (count) the number of white blood cells using an automatic blood analyzer, first, a red blood cell lysing agent containing a surfactant, etc. is added to the blood sample to selectively lyse the red blood cells in the blood sample. The remaining white blood cell measurement sample is prepared, and then the white blood cell measurement sample is passed through a narrow passage or pore provided in the detection section of the home-made blood analyzer. This is done by detecting an electrical or optical signal generated by a detection section.

Furthermore, in recent years, improvements have been made to red blood cell lysing agents to minimize the deformation of white blood cells, and devices that classify and count white blood cells into classes such as granulocytes, monocytes, and lymphocytes based on the difference in signal intensity obtained have been introduced. It has been realized. If this automatic blood analyzer is used, white blood cells can be classified and counted much more easily than the visual counting method described above.

On the other hand, hemoglobin is usually measured using the cyanmethemoglobin method. In the cyanmethemoglobin method, a red blood cell lysing agent containing a nonionic surfactant or the like is added to a blood sample to elute the hemoglobin contained in the red blood cells, and then the hemoglobin that is evaporated is oxidized by the action of an oxidizing agent such as potassium 7-erythyanide. Oxidize to form methemoglobin, and finally add cyanide ions to convert methemoglobin to cyanmethemoglobin (BiCI).
W) A stable sample for hemoglobin measurement is prepared by: Absorbance '4r of the prepared sample for hemoglobin measurement at a specific rBL length: Hemoglobin lI[ is measured by the measuring method. This method is recognized as an international standard method. Although the sample prepared by this method is a very stable substance, since the oxidizing agent is removed to oxidize the hemoglobin, it takes several minutes for the oxidation to be completed. 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 eliminate hemoglobin. 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, it is dangerous to handle it, and the waste liquid after measurement must be disposed of after decomposing the cyanide using sodium hypochlorite, etc. There is,
Waste liquid treatment is troublesome.

Therefore, red blood cells can be lysed using only a nonionic surfactant without being oxidized to methemoglobin, which is hemoglobin.
A method (oxyhemoglobin method) in which oxyhemoglobin (EbO,) K is converted and the absorbance at a specific wavelength is measured has also been used. 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 oxyhemoglobin method has the problem that blood samples with high methemoglobin content cannot be measured correctly. This is because methemoglobin is not converted to oxyhemoglobin. RIK' as an example:
1 shows comparative data of measurement results between the oxyhemoglobin method and Example 1 of the present invention when the methemoglobin content is varied using I.trol blood. Control blood is used as a substance for quality control in automatic blood analyzers, and is usually stored refrigerated and shows stable hemoglobin values over a long period of time.However, if stored above room temperature, the hemoglobin in the blood gradually changes to methemoglobin. It turns into Therefore, as shown in Table 1, when measuring control blood stored at 22°C using the oxyhemoglobin method, if the hemoglobin method is used, the amount of hemoglobin converted to methemoglobin will be measured. After several days, the &II constant value gradually becomes lower than the initial value.

As a way to solve this problem, Ohe et al. proposed that sodium dodecyl sulfate or sodium lauryl sulfate (SLS) (anionic surfactant) and tritone in a neutral 11@ solution (F#7.2) were added in equal amounts. A reagent for hemoglobin measurement containing X-100 (nonionic surfactant) was developed by Clinical Biochemistry (CLis,
Bitschatm, ) 15, 83 (1982). In this method, red blood cells are lysed by the action of SLS and Triton X-100, and the eluted hemoglobin 19I:Methemoglobin is simultaneously converted to SLS hemoglobin by the action of SLS. With this method, the hemoglobin concentration in the blood can be measured without being affected by methemoglobin, and since it does not contain cyanide, there is no need to dispose of waste liquid.However, with this method,
White blood cells cannot be measured at the same time as hemoglobin measurement. Also, Japanese Patent Publication No. 1986-14 s a
Although a reagent for measuring hemoglobin using an ionic surfactant has been published in ``69 I-cyanide-free hemoglobin reagent'', it is not possible to measure white blood cells at the same time as hemoglobin measurement even with this reagent.

As described above, in the conventional technology, E[
To measure white blood cells at the same time as hemoglobin measurement,
Furthermore, it was not possible to correctly measure hemoglobin in a blood sample with high methemoglobin content.

Table 1 Control blood - fixed pot (oxyhemoglobin method F5 implementation fPIjl) initial value 16
．． OF// 17. Ojl/122℃, 3 days storage
15.7f/l 17.017 Stored at 122°C for 7 days 15.5P/l 17. OpH The present inventor has developed a reagent to solve these problems by patent application No. 1-2752.
I applied earlier as 80 years old. By strictly specifying the type and concentration of Kabuon surfactant, non-ionic surfactant, amphoteric surfactant, or oxidizing agent used as a red blood cell lysing agent, hemoglobin elution can be achieved almost instantaneously. This reagent makes it possible to measure hemoglobin by converting it into meth, and also makes it possible to measure white blood cells at the same time. However, since this reagent does not contain a component that stabilizes methemoglobin, there is a problem in that the hemoglobin value changes slightly over time after the measurement sample is prepared, as shown in Comparative Example 2 in Table 2.

This is a fully automatic type sample v! In the case of an apparatus that automatically performs the 4-manufacturing and measurement, the time required for the electrical manufacturing measurement after the preparation of the sample liI is short and strictly regulated, so this does not pose a problem and does not negate the effects of the previous invention.

- Currently, in order to reduce the cost of measurement equipment, semi-automatic equipment is also used in the market, in which a person prepares the sample and the equipment only performs the measurement.

In a semi-automatic type i-position, the time from sample preparation to measurement is irregular, and usually there is a variation of several minutes to several tens of minutes from sample preparation to measurement.

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

As described above, with the conventional technology, it is possible to measure white blood cells at the same time as hemoglobin using a reagent that does not contain cyanide, to accurately measure hemoglobin in a blood sample with a high methemoglobin content, and to obtain a sample for hemoglobin measurement that is stable over a long period of time. I couldn't get it.

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

That is, a reagent for counting leukocytes in blood and measuring hemoglobin concentration that satisfies the following conditions,
This reagent is characterized by the fact that it does not contain cyanide and keeps hemoglobin stable for a long period of time.

l] A quaternary ammonium salt (structure a) at a concentration that can hemolyze red blood cells in the blood and oxidize hemoglobin, or
at least a cationic surfactant selected from the group consisting of compositions of pyridinium salts (Structure 6);

Structure α Structure C II) R,: C, ~C1° alkyl group, alkenyl group, or alkynyl group, R1 ~ R,: C, ~C, alkyl group, alkenyl group, or alkynyl group, X-: anionic group Structure b An integer of % near-19, X-: @ionic group Contains at least one member selected from the group consisting of cationic, nonionic, and amphoteric surfactants having the structure C--.

R,:C, ~C1° alkyl group, alkenyl group, or alkynyl group, R2~/? , :C, ~Cs alkyl group, alkenyl group,
Or alkynyl group . Alkyl, alkenyl, or alkynyl group? l: Integer structure of 10 to 50 - to R, :C, ~C3゜alkyl group R1 to R, :C, ~Ca alkyl group, alkenyl group, or alkynyl group consisting of a hemoglobin stabilizer shown in ~n) Contains at least ls selected from the group and its concentration is f) ~ n
).

f) Tyrone Concentration 0.1-1000 Da/! k) Bipyridine Concentration lO~10000■/<) 1.10-Phenanthroline and the day conductor Concentration lO~3000η/j Q) 8-Hydroxyquinoline Concentration 0.1R,,R
, : -H, -CH.

X, I 7 (snee tl, phenyl group, -○H.

j) Phenol compound concentration 0.1-1000Q/1 10001111i/J v*) Phenyl 5-pyrazolone and its derivatives concentration 0.1-10000η/l 3 R1: -H, -C OH, -CH0%-COOH ,−
0HR, Knee H, -COON, -0HR R, Knee H, -COOH k) Hiss 7 x / -# A Concentration 0.1 to 1
000119/1 R3, R,: -M or C1 to C4 lower alkyl group -B) 7x=3-pyrazolone 1111 0.1 to 50,000 ■/E L) Pyrazole and its derivatives Concentration L1 .1~100
00~/j R,,R,,J? , : -H or lower alkyl group of CI to C6, +v) The solution has a pH range of 3.0 to 9.0.

V) The total concentration of the quaternary ammonium salt is 0.1 to x
Must be within the range of s, oy/j.

vl) The pyridinium salt am degree is 0.1 to 15
．． Must be within the range of 0P/j.

V+D The total concentration of the nonionic surfactant is 0.1
~ls,oy/j.

vifD The total envy of the amphoteric surfactant is 0.1 to 1
Must be within the range of 5.0P/l.

1x) The total concentration of the cationic surfactant is 0.1 to 1
5. Must be within the range of OF/l.

By appropriately combining components α) to #) in the above reagent, leukocytes can be fractionated into two or three types, such as lymphocytes; monocytes, eosinophils, basophils; and neutrophils. .

In general, hemoglobin measurement is performed by measuring hemoglobin in red blood cells by using an excessive red blood cell lysing agent, as described above.
The heme iron in the eluted hemoglobin is oxidized by the action of a red blood cell lysing agent and oxygen in the solution, or by the action of an appropriate oxidizing agent, and the resulting methemoglobin is treated with a conventional methemoglobin stabilizer such as cyanide ions. This is done by stabilizing the hemoglobin sample by acting on it and measuring the absorbance of the stabilized hemoglobin sample. However, the presence of this conventional methemoglobin stabilizer also causes the waste liquid treatment problems mentioned above. Methemoglobin that does not contain a methemoglobin stabilizer is more unstable over time than the aforementioned cyanmethemoglobin, oxyhemoglobin, etc., and its absorbance changes depending on the pH of the solution, making it difficult to measure hemoglobin. was not used. The present inventors previously created a reagent in Japanese Patent Application No. 1-275280 for stabilizing methemoglobin without containing the above-mentioned methemoglobin stabilizer. In this reagent, first, red blood cells in a blood sample are selectively lysed mainly by the action of a quaternary ammonium salt or a pyridinium salt, and hemoglobin in the red blood cells is eluted. The eluted hemoglobin undergoes almost instantaneous denaturation (change in stylistic structure), and the heme iron in hemoglobin is oxidized from divalent to trivalent to methemoglobin by the oxygen dissolved in the reagent.

When a single type of cationic surfactant is used,
This reagent has the same problems as methemoglobin prepared by the method described above, but it also has cations, nonions,
By adding an appropriate amount of an amphoteric surfactant or the like, the degree of denaturation of hemoglobin can be adjusted and stable methemoglobin can be obtained. Furthermore, the negative effect can be overcome by adding a buffer as described below. The mechanism of action for stabilizing methemoglobin is not clear, but it is probably due to the action of multiple surfactants with different molecular structures on hemoglobin that the degree of denaturation (change in steric structure) to methemoglobin can be kept constant. This is thought to have the effect of fixing it at the level of . The reagent disclosed in Japanese Patent Application No. 1-275280 does not destroy white blood cells either (because they remain, it is possible to measure the white blood cell count).

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

The effects of hemoglobin stabilizers are shown in Table 2.

Although the mechanism of action of the hemoglobin-7 stabilizer of the present invention is not clear, it is likely that (in the molecular structure of the hemoglobin stabilizer, an arc electron pair of a nitrogen atom or an oxygen atom in a phenolic hydroxyl group) is thought to bind to heme iron in methemoglobin as a chelate, thereby stabilizing methemoglobin.

With this reagent, it is possible to fractionate leukocytes into two or three groups by specifying the surfactant concentration more strictly.

An example of the composition of the reagent of the present invention is shown below.

Composition example 1 Concentration range of tyron 0.1 to IUOM Sodium chloride composition example 2 1.0 to IO, OF l Concentration range of salt tutyron u, t to 1 ouomg Phosphate buffer 1/15 to 1/6 ON Sodium chloride 1.0-10. OF Purified water 1 Composition example 3. d1 degree range lauryltrimethylammonium 0.3~io,
oy dichloride (quaternary ammonium salt ncetyltrimethylammonium salt 0.01-2.
OF Roride (&!! Quaternary ammonium salt) Tyrone
0.1~1000~Phosphate buffer 1715~760M Sodium chloride 1.0~lo
, oj'M Seisui 14
In composition examples 1 to 3, red blood cells are hemolyzed by the action of a quaternary amnium salt and a surfactant such as an amphoteric surfactant, and the eluted hemoglobin is converted to meth and stabilized, and further stabilized for a long time at °C by the action of Tyron. This is what I did.

Composition Example 1 uses a combination of a quaternary ammonium salt and an amphoteric surfactant to enable the measurement of leukocytes, while Composition Example 2 uses a combination of a quaternary ammonium salt and a nonionic surfactant to measure at least two leukocytes. In composition example 3, 21'! By combining quaternary ammonium salts of the same class, leukocytes can be fractionated into at least three types.

In the case of Composition Example 3, the total concentration of quaternary ammonium salts described in the claims is 0.31 to 12.0, which is the sum of the concentration of lauryltrimethylammonium chloride and the concentration of cetyltrimethylammonium chloride. O
It means the concentration range of j'.

Composition Example 4 #Ii Degree Range Lauryl Trimethyl Ammonia 1.0-10. O
F muchloride phorioxyethylene nonyl 72 1.0-10. O
j' Nyl ether 8-hydroxyquinoline 0.1A-1000 ~ Phosphate buffer 1715 ~ 760M Sodium chloride 1.0 ~ lo, OF Purified water t Composition example 5 Lauryl trimethyl ammonium chloride concentration range 3.0 ~ lt1. OF Sexual agent) 1.10-7 enanthroline) 10-3000- Phosphate buffer 1715-760M Sodium chloride 1.0-IO, Oj' Purified water J Composition example 6 Laurylpyridinium chloride (pyridinium salt) Concentration range 0. 5-1U, OP lauryl dimeralamine acetate 0.5-10. O
F main 1~Phenyl 3-pyrazolone 0,1~10000~
Phosphate buffer 1715-720M Sodium chloride 1.0-1O0
0! lI water 1
j Composition Example 4 is a combination of a quaternary ammonium salt, a nonionic surfactant, and 8-hydroxyquinoline, and Composition Example 5 is a combination of a quaternary ammonium salt, a cationic surfactant, and IO-7. In combination composition example 6 with enanthroline, hemoglobin is memethized and stabilized by the combination of a quaternary ammonium salt, a pyridinium salt, and 1-phenyl-3-pyrazolone, and further, hemoglobin is stabilized for a long time by the action of a hemoglobin stabilizer. It has been stabilized.

In addition, it is also possible to fractionate leukocytes into two or more populations by adjusting the concentration of each surfactant. In composition examples 1 to 6, the phosphate buffer is pH within the range of 3.0 to 9.0
Any suitable buffer such as citric acid, maleic acid, Tris, etc. may be used (not limited to phosphoric acid. Furthermore, The preferred pH range is 5.0 to 8.0.

When the pH becomes 3.0 or less, damage to white blood cells becomes large, making it difficult to measure white blood cells. In addition, if the pH exceeds 9.0, the stability of hemoglobin over time deteriorates (it becomes chlorinated). Any inorganic or organic salt may be used as long as it dissociates into ions.

The concentrations of each component indicated in the claims indicate the concentrations in a state where it can be measured simply by adding blood to the reagent without the need for other diluents, etc. When used in a conventional device that measures hemoglobin or white blood cells by mixing a diluent and a lysis reagent at a fixed ratio, the concentration of each surfactant and other components must be changed according to the mixing ratio of the device. It is also possible. In that case, the concentration of each component shown in the claims represents 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; 1) blood samples with high hemoglobin content can also be measured; +V) A reagent with long-term hemoglobin stability can be supplied.

Furthermore, when this reagent is used in an automatic blood analyzer, there is no need to wait for the sample preparation section for hemoglobin measurement and the sample preparation section for white blood cell measurement separately, resulting in a simpler device and lower cost.

〔Example〕

The effect when a reagent #14 having the following composition was prepared and the hemoglobin stabilizer shown in Table 2 was added is shown.

Concentration Lauryltritylammonium chloride 1.25F Lauryldimethylaminoacetic acid betaine 1.00y Purified water J The amount of change in hemoglobin and the rate of change in Table 2 were calculated using the following formula.

Hemoglobin change - Hemoglobin measurement value 30 minutes after sample preparation - 2 after sample preparation
Hemoglobin measurement value at 0 seconds Single change in hemoglobin (hemoglobin change/sample preparation & hemoglobin measurement value at 20 seconds) XIOIJ All measurement devices are F-800 (manufactured by Toa Medical Electronics Co., Ltd.)
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, as shown in Table 2, the hemoglobin value changed from sample preparation vk20 seconds. The thickness increases (changes) within 30 minutes. Comparative Example 2 is an automated cyanmethemoglobin method used in an automatic blood analyzer,
The amount of change is sufficiently small.

Comparative Example 3 shows a typical example of hemoglobin in which methemoglobin was prepared using a single surfactant (lauryltrimethylammonium chloride) except for betaine lauryldimethylaminoacetate in the above composition. Because methemoglobin is not stabilized, the hemoglobin value increases (changes) between 20 seconds and 30 minutes after sample preparation.

In Comparative Example 4 (composition example of Japanese Patent Application No. 1-275280), in which an amphoteric surfactant, lauryldimethylaminoacetic acid betaine, was further added to the composition of Comparative Example 3, the amount of change is reduced compared to Comparative Example 3.

Furthermore, in the compositions (Examples 1 to 9) in which a hemoglobin stabilizer, which is a reagent of the present invention, was added, the amount of change was further reduced compared to Comparative Example 4.

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 Fukua Medical Electronics Co., Ltd.) and the results of measuring the number of white blood cells using the reagent of Example 1.

Correlation coefficient r-0.999. Regression straight 11y-1,009m
It shows a very high correlation of -0,179.

FIG. 2 shows a correlation 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.

Correlation coefficient γ-0,999, regression line C 1.011g-
It shows a very high correlation with 0°019.

As shown in Table 1, the amount of methemoglobin increased,
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.

The reason why there is a difference between the initial values determined by the oxyhemoglobin method in Table 1 and the initial values determined by the reagent in Example 1 is because control blood was used in which a certain amount of hemoglobin in the dish was converted to methemoglobin from the beginning. It is.

Implementation Ga 10゜ A reagent with the following composition was prepared.

Concentration lauryl trityl ammonium 1.505'
Chloride Cetyl Toriderammonitum Chloride 0.403' Tyrone Purified Water Implementation f1111° A reagent with the following composition was prepared &14.

3 C1OmLg J Ufy Li/Iz Triple ammonium chloride concentration 3.20P Cetyl tridel ammonium chloride 0.20F Tyron purified water 300~ When using the reagents of Example 1.1O111 in Figures 3 to 5 of 1, respectively Figure 2 shows the white blood cell particle size distribution. In Figures 3 to 5,
The vertical axis represents the relative frequency of blood cells, and the horizontal axis represents the relative intensity of the blood cell signal (ie, the size of the blood cells) obtained when blood cells are measured by an automatic blood analyzer. When the reagent of Example 1 is used, leukocytes exhibit a unimodal particle size distribution as shown in FIG. When using the reagent of Example 1O,
As shown in FIG. 4, white blood cells exhibit 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. When the reagent of Example 11 was used, the leukocytes showed a particle size distribution divided into three areas: area A, area C, and area C, 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, in addition to being able to obtain the same measured values as conventional methods, 1) hemoglobin and leukocytes can be measured in one sample, and leukocytes can also be fractionated.

++) Me) Blood samples with high hemoglobin content can be measured correctly.

:11) Since it does not contain toxic substances such as cyanide, there is no need for waste liquid treatment.

IV) A reagent with long-term hemoglobin stability can be supplied.

[Brief explanation of drawings]

Figure 1 shows the white blood cell count measurement results obtained using the conventional method and F-SOU (manufactured by Toa Medical Electronics Co., Ltd.)
Leukocytes with 1111 reagents! It is a correlation diagram with measurement results. FIG. 2 is a correlation diagram between the hemoglobin value measurement result using the conventional method [fan methemoglobin method (international standard method)] and the hemoglobin value measurement result using the reagent of Example 1. 3 to 5 are graphs showing the leukocyte particle size distribution when using the reagents of Examples 1 and 10.11, respectively. In FIGS. 3 to 5, the vertical axis represents the relative frequency of blood cells, and the horizontal axis represents the relative intensity of the blood cell signal (i.e., the size of the blood cells) obtained when blood cells are measured by an automatic blood analyzer. In FIG. 3, leukocytes exhibit 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. The white blood cells shown in FIG. 5 have a particle size distribution divided into three regions: A, C, and A. 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. q# Applicant Toa Medical Electronics Co., Ltd. (4 others) 1. Indication of the case Patent Application No. 50813 of 1990 2. Name of the invention Toa Medical Electronics Co., Ltd. 4. Agent 11) Claim for patent Correct the range as follows. 1. A reagent for counting leukocytes in blood and measuring hemoglobin concentration that satisfies the following conditions, and is characterized by not containing cyanide and keeping hemoglobin stable for a long time. ) A cationic surfactant selected from the group consisting 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. Contains at least one type. Structure a R1: 08-C2o alkyl group, alkenyl group, or alkynyl group ・R2-R4: C□-08 alkyl group, alkenyl group, or alkynyl group. X-: Anionic group Structure b R1: C8-C2o alkyl group, alkenyl group, or alkynyl group. n integer of 19, x-: anionic group 11) A cation or nonion having the structure ce. From the group consisting of amphoteric surfactants. Contains at least one selected type.Structure CR: 08~C,!o Alkyl group, alkenyl group. Or alkynyl group. R2νR3: C1~08 alkyl group, alkenyl group, or alkynyl group. Xl: Anion Group structure d R□-R2-(CH20H20)n-Hn: 10-50
Integer structure e R1: 08-020 alkyl group, R2-R3: C1-C8 alkyl group, alkenyl group, or alkynyl group 11i) selected from the group consisting of hemoglobin stabilizers shown in f) to n) Contains at least one type and its concentration is as shown in f) to n). f) lian, 114 degrees 0.1~1000 IIT
9/zR2, R3 knee H, phenyl group, -0H1j) Phenolic compound, S degree 0.1-1000η/1 g] 8-Hydroxyquinoline, # degree 0.1-11000
In/1 H h) Bipyridine, concentrated [10-10000iv/zi)
1.10-phenanthroline and its derivatives, concentrated 1i
10~300011g/lR1 knee H1~C)I 20
H1~cHo, -cooH1OH R2 knee H2-coo9! J, -0g(R3 knee H,
-COOH k) Hiss 7ff-/-le A, 8 degrees 0.1-100
0IRy/l l] Pyrazole and its derivatives, # degree o, 1 to ic+
oooIv, "z R1, R4: -H, -CH3 R□, R2, R3 nee H or lower alkyl group of 01 to 04. m] Phenyl 5-pyrazolone and its derivatives, concentration 0
．． 1 to 10,000 da/l RRR--H or C□ to C4 lower grade 1' 2
'3' Alkyl group. n) Phenyl 6-pyrazolone, 8 degrees 0.1-10000
In9/4 1) The solution has a pH in the range of 3.0 to 90. (2) The total concentration of the quaternary ammonium salt is 0.1 to 15.
Must be in the range of Ot/l. Vi) The total concentration of the pyridinium salt is 0.1 to 15
．． Must be within the range of 05'/l. vlo The total concentration of nonionic surfactants is 0.1
~15. (Being in the range of 1#. vm) The total concentration of the amphoteric surfactant is 0.1 to 15.0.
Must be within the range of 5'/l. 1x) the total concentration of the cationic surfactant. 0.1-15. Must be in the Off/l range. 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. 2. The reagent of claim 1, which is in the range of #Of# and can be fractionated into at least two leukocytes. 6. Contains at least one type selected from the group consisting of quaternary ammonium salts, the total degree of the quaternary ammonium salts is in the range of 0.1 to 12.0), and has nonionic specific surface activity. The total concentration of nonionic specific surfactants is 0.1 to 12.
The reagent according to claim 1, which is in the range of 0%/l and capable of fractionating leukocytes into at least two types, contains at least one kind selected from the group consisting of quaternary ammonium salts, and is capable of fractionating leukocytes into at least two types. The total concentration of ammonium salts is in the range of 0.1 to 12.0 P/l and contains at least one type selected from the group consisting of diampholytic surfactants;
The total concentration of amphoteric surfactants ranges from 0.1 to 12.05'/l. The reagent according to claim 1, which is capable of fractionating leukocytes into at least two types. 5. Contains at least one type selected from the group consisting of quaternary ammonium salts, and has a total concentration of quaternary ammonium salts of 0.1 to 12.0? Contains at least one type selected from the group consisting of cationic surfactants, and the total concentration of cationic surfactants is 0.1 to 5.0P.
2. The reagent according to claim 1, which is in the range of /l and is capable of fractionating leukocytes into at least two types. 6. Contains at least one type selected from the group consisting of quaternary ammonium salts, and the total concentration of quaternary ammonium salts is 0.1 to 12. (within the range of 1/l and containing at least one type selected from the group consisting of pyridinium salts,
The total concentration of pyridinium salts is in the range of 0.6 to 10.0 P/l. The reagent according to claim 1, which is capable of fractionating leukocytes into at least two types. Z Contains at least two types selected from the group consisting of quaternary ammonium salts, and the total concentration of quaternary ammonium salts is 2.2 to 5. 2. The reagent according to claim 1, which is in the Off/l range and can be fractionated into three leukocytes. 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 3.0-12.0 t/l, and nonionic and fruit surface active notes. Contains at least one type selected from the group consisting of nonionic surfactants, and the total concentration of the nonionic surfactant injection agent is 1.0-12
．． The reagent according to claim 1, which is in the range of 0 P/l and is capable of fractionating leukocytes into six groups. 9 At least one type selected from the group consisting of quaternary ammonium salts? containing, and the total concentration of quaternary ammonium salt is 1.0 to 10. Contains at least one type selected from the group consisting of C1/4 and amphoteric surfactants.
The total concentration of amphoteric surfactants ranges from 0.1 to 10.0 P/l. The reagent according to claim 1, which can be fractionated into six leukocytes. 10. 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 1.0 to 10.05'/l, and the total concentration of quaternary ammonium salts is in the range of 1.0 to 10.05'/l, and The total concentration of pyridinium salts is 1.0 to 5. The reagent according to claim 1, which has an OF/l range and is capable of fractionating leukocytes into three groups. ” +2+ Correct the details of 8A as shown below. Page Line Wrong Correct 14
14 Hemoglobin...Hemoglobin Hemoglobin is 17 4 2
431 12 Phenanthroline] Phenanthroline 32 1 Methyl Methyl 3
212-13 Quaternary ammonium amphoteric surfactant salt (3) The formula on page 24, line 6 of the specification is corrected as follows. (4) Correct as shown in Figure 5, attached. or more” (relative*2)

Claims (1)

[Claims] 1. A reagent for counting leukocytes in blood and measuring hemoglobin concentration that satisfies the following conditions, and is characterized by not 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.▼] ^-: Anionic group structure b ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ n: An integer from 7 to 19, X^-: Anionic group ii) From cationic, nonionic, and amphoteric surfactants with structures c to e Contain at least one type selected from the group consisting of: Structure c [▲There are mathematical formulas, chemical formulas, tables, etc.▼] ^-: Anionic group 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. Yes ▼ 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 hemoglobin stabilizer selected from the group consisting of f) to n). Contains the types, and the concentration thereof is as shown in f) to n). f) Tyron, concentration 0.1-1000mg/l▲mathematical formula,
There are chemical formulas, tables, etc. ▼ g) 8-Hydroxyquinoline, concentration 0.1-1000m
g/l ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ h) Pipyridine, concentration 10 to 10,000 mg/l ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ i) 1,10-phenanthroline and its derivatives, concentration 10 to 3,000 mg /l ▲There are mathematical formulas, chemical formulas, tables, etc.▼ R_1, R_4: -H, -CH_3 R_2, R_3: -H, phenyl group, -OH, j) Phenol compound, concentration 0.1-1000mg/l ▲Mathical formula , chemical formulas, tables, etc. ▼ R_1: -H, -CH_3OH, -CBO, -COOH
, -OHR_2: -H, -COOH, -OH R_3: -H, -COOH k) Bisphenol A, concentration 0.1-1000mg/l ▲There are mathematical formulas, chemical formulas, tables, etc.▼, l) Pyrazole and its derivatives, Concentration 0.1-1000
0mg/l ▲There are mathematical formulas, chemical formulas, tables, etc.▼ R_1, R_2, R_3: -H or C_1 to C_4
lower alkyl group, m) phenyl 5-pyrazolone and its derivatives concentration 0.1
~10000mg/l ▲There are mathematical formulas, chemical formulas, tables, etc.▼ R_1, R_2, R_3: -H or C_1 to C_4
lower alkyl group, n) phenyl 3-pyrazolone, concentration 0.1-10000
mg/l ▲There are mathematical formulas, chemical formulas, tables, etc.▼ iv) The solution must 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. 3. Contains at least one type selected from the group consisting of quaternary ammonium salts, has a total concentration of quaternary ammonium salts in the range of 0.1 to 12.0 g/l, and is composed 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 type selected from
2. The reagent of claim 1, wherein the total concentration of pyridinium salts is in the range 0.3 to 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 three 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 3.0 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.