CN1277125C - Cyanide-free hemolysin - Google Patents

Abstract

The present invention relates to a hemolysin composition and a use method thereof for measuring total hemoglobin concentration(manually or automatically) in a blood sample and is used for simultaneously counting the number of leucocytes in the blood sample and counting the number of leucocyte subgroups in a classifying mode. A reliable method is provided for measuring the hemolysin of haemoglobin, and simultaneously, leucocytes can be divided into at least two subgroups for being respectively counted. The other goal is to provide the method of using the hemolysin. The hemolysin without cyanide has the technical scheme comprising at least one kind of surface active agent group which is sufficient for dissolving erythrocytes and releasing the amount of haemoglobin, at least one kind of cationic surface active agent, at least one kind of anionic surface active agent and at least one kind of non-ionic surface active agent. By adopting the scheme, the situation that haemoglobin can be measured by using hemolysin containing no cyanide and having no toxicity and by a reliable method, and simultaneously, leucocytes can be divided into at least two subgroups for being counted respectively.

Description

Cyanide-free hemolysin

technical field

The present invention relates to a hemolysin composition for determining (manually or automatically) total hemoglobin concentration in a blood sample and for simultaneously performing leukocyte count or differential count of leukocyte subsets.

Background technique

The determination of total hemoglobin is an important indicator of blood oxygen carrying capacity. So far, more than 300 kinds of hemoglobin have been found in normal people and patients with blood diseases. These abnormalities lead to changes in hemoglobin concentration or changes in the oxygen-carrying capacity of hemoglobin, resulting in diseases such as sickle cell anemia, thalassemia and hemoglobin abnormalities.

Determining the concentration of hemoglobin in blood samples is a very important basis for the diagnosis of related diseases, and it is also very important for monitoring the treatment of hemoglobin diseases and other diseases that may affect the change of hemoglobin levels.

In analytical experiments for the manual or automatic determination of the total hemoglobin concentration in blood samples and for simultaneous white blood cell counts or differential counts of white blood cell subsets, although the chromogen formed by the standard cyanide hemoglobin method and its modified automatic method Stable and reliable, however, due to the use of potassium cyanide, reagent waste will cause great environmental pollution. In the past decade, a great effort has been devoted to the development of an automated hemoglobin analysis method that does not use cyanide.

Oshiro et al. [Clinical Biochemistry (Clin. Biochem.), 1583 (1982)] describe the reagents used for the analysis of hemoglobin, which include sodium lauryl sulfate (SLS) and TritonX-100 (non-ionic surface active agent). SLS is used to lyse red blood cells and is thought to further generate the SLS-hemoglobin complex, which has an absorbance maximum at 539 nm and a shoulder at 572 nm. The SLS method is now recognized as a safe and reliable cyanide-free method, but it is impossible to analyze white blood cells at the same time as the hemoglobin measurement using the oshiro method. Because the high concentration of sodium lauryl sulfate will destroy the white blood cells too deeply.

US Patent No. 5,242,832 (genus Sakata) discloses cyanide-free hemolysin for counting white blood cells and determining hemoglobin concentration in a blood sample. The hemolysin comprises at least one surfactant including cationic surfactant and amphoteric surfactant and at least one hemoglobin stabilizer selected from the following compounds: Tiron, 8-hydroxyquinoline, bipyridine, 1,10-phenanthrene Phenols, phenolic compounds, bisphenols, etc. Sakata demonstrated that leukocyte fractionation or three groups (including lymphocytes, aggregates of monocytes, eosinophils and basophils, and aggregates of neutrophils) can only be achieved by using at least two suitable surfactants and strict This can only be accomplished by controlling the surfactant concentration. Sakata also stated that the preferred pH value of hemolysin is 5.0-8.0. If the pH is 3.0 or less, damage to leukocytes increases, thus making measurement of leukocytes difficult, and if the pH is 9.0 or more, the stability of hemoglobin deteriorates over time.

PCT/US95/02897 (Kim) discloses a cyanide-free method and reagents for the determination of hemoglobin in whole blood samples. The reagent includes a ligand (selected from the following compounds: imidazole and its derivatives, N-glycolamide, H-hydroxylamine, pyridine, oxazole, thiazole, pyrazole, pyrimidine, purine, quinoline and isoquinoline) and a A surfactant (selected from dodecyldimethylamine oxide and octylphenoxypolyethoxyethanol) with strong erythrocyte lysing ability. The analysis method is fast, less than 10 seconds. However, this reagent only operates at an extremely alkaline pH of 11-14. In addition, Kim does not address the ability to count leukocytes or separate leukocyte subpopulations.

None of the cyanide-free hemoglobin assays described above provide monocyte and whole granulocyte counts at the same time as hemoglobin; however, both parameters are valuable tools for clinical diagnosis of various diseases . There is a need for more versatile cyanide-free hemoglobin assays and multifunctional reagents that can perform several diagnostic assays in a single automated step.

Chinese patent application No. 98801947 belongs to Kurt Corporation, which discloses a cyanide-free reagent composition, which contains hemoglobin ligand triazole and derivatives thereof, tetrazole and derivatives thereof, 4,6- Alkali metal salts of dihydroxy-1,3,5-triazine-2-carboxylic acid, melamine, aniline-2-sulfonic acid, quinalidic acid, 2-amino-1,3,4-thiadiazole, tri Oxazine and its derivatives, urazole, DL-2-piperidine, isonicotinamide, anthranil, 6-aza-2-thiothymine, adenine, 3-(2-thienyl) Aqueous solution of organic ligands of acrylic acid, benzoic acid and alkali metal and ammonium salts of benzoic acid, pyrazine and its derivatives. There are also hemolytic surfactants selected from quaternary ammonium salts, pyridinium salts, organophosphates and alkylsulfonic acids to lyse red blood cells to release hemoglobin. However, this method still uses certain toxic or carcinogenic compounds, which still pollutes the environment to a certain extent.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned defects of hemolysin, to provide a hemolysin which does not contain cyanide, is non-toxic, can adopt a reliable method to measure hemoglobin, and at the same time leukocytes can be divided into at least two subpopulations for separate Calculate.

The technical solution of the present invention is that the described cyanide-free hemolysin contains an anionic surfactant that can coordinate with hemoglobin, selected from alkyl sulfonate, alkylbenzene sulfonate and alkyl sulfate, and an anionic surfactant that can dissolve red blood cells The cationic surfactant is selected from quaternary ammonium salts, pyridinium salts, amine oxide type quaternary ammonium salts, and non-ionic surfactants for solubilization. This composition can be used over a wide pH range.

The further form of the technical solution of the cyanide-free hemolysin of the present invention is at least one surfactant in an amount sufficient to dissolve red blood cells and release a certain amount of hemoglobin, and the surfactant is selected from the group consisting of the following substances:

1) At least one cationic surfactant, which is capable of lysing all human red blood cells and dividing white blood cells into two subpopulations with a specific instrument within a suitable concentration range. Including quaternary ammonium salt, pyridinium salt, amine oxide quaternary ammonium salt.

R1 and R5 in the formula are long-chain alkyl, alkenyl or alkynyl groups with 8-18 carbon atoms, and R2, R3 and R4 are all short-chain alkyl, alkenyl, or alkynyl groups with 1-5 carbon atoms. group or hydrogen atom, x- is any kind of anion.

2) At least one anionic surfactant can combine with hemoglobin to form a chromogen at a suitable concentration, thereby measuring its concentration. The anionic surfactant has the following structure:

R ₁ -SO ₄ ^{-X +}

In the formula, R1 is a long-chain alkyl, alkenyl or alkynyl group with 8-18 carbon atoms, and x+ is any alkali metal cation or ammonium ion.

3) at least one nonionic surfactant having the following structure:

Wherein R1 is a long-chain alkyl group, alkenyl group or alkynyl group with 6-8 carbon atoms, and n is an integer of 4-40.

In a preferred form, the combination can be used to simultaneously provide a determination of total hemoglobin concentration, a count of leukocytes and a separate count of leukocyte subpopulations in a blood sample, wherein said leukocytes are differentiated to include lymphocytes, monocytes and granulocytes of the three subgroups.

Among the cationic surfactants for lysing red blood cells, quaternary ammonium salts are preferred. The concentration of surfactant in the hemolysin composition needs to be sufficient to lyse red blood cells and release hemoglobin while preserving the white blood cell nuclei. In order to count leukocytes, it is not necessary to leave the leukocyte membrane intact. In general, when the hemolytic surfactant in the hemolysin composition of the present invention described above is used, the white blood cell membrane is partially lysed while the red blood cells are completely lysed and destroyed. The nuclei left by the leukocytes allow the counting of leukocytes and the separation of leukocyte subpopulations into lymphocytes, monocytes, and granulocytes using the DC impedance method.

The cationic surfactant concentration in the above hemolysin composition is about 2 g/L to about 100 g/L, preferably about 15 g/L to about 60 g/L.

The concentration of anionic surfactant in hemolysin is sufficient to allow all hemoglobin to form an anionic surfactant-hemoglobin complex. If the amount of anionic surfactant is insufficient, the chromogen of the formed complex is destabilized.

The concentration of the anionic surfactant in the cyanide-free hemolysin of the present invention is in the range of 0.1-10 g/L, preferably 0.5-3 g/L, which is most suitable.

The invention has no cyanide hemolysin, and the concentration range of the nonionic surfactant can be in the range of 0.5-5g/L.

In the present invention without cyanide hemolysin, the concentration of the cationic surfactant can be 25g/L, the concentration of the anionic surfactant can be 1.5g/L, and the concentration of the nonionic surfactant can be 0.58g/L.

Cyanide-free hemolysin of the present invention, described cationic surfactant is dodecyltrimethylammonium bromide, and its concentration is 25g/L, and anionic surfactant is sodium lauryl sulfate (SLS), and its concentration is 1.5 g/L, nonionic surfactant is emulsifier OP, its concentration is 0.58g/L.

The present invention provides a method of using the hemolysin, which is used in conjunction with appropriate blood thinners to measure the hemoglobin concentration in the blood or to simultaneously count white blood cells or separately count white blood cell subgroups.

The method comprises diluting a blood sample with a suitable blood thinner, mixing a sufficient amount of a hemolysin composition with the diluted sample, measuring the sample photometrically at a wavelength predetermined for the determination of hemoglobin concentration, while The leukocytes are counted or at least two subsets of the leukocytes are counted separately on the hematology analyzer of the measuring device.

Dilute the anticoagulated blood or capillary blood with a suitable diluent, mix the appropriate amount of the above-mentioned hemolysin with the diluted sample (manually or automatically by the instrument), and the dilution ratio is from 125:1 (total reagent volume relative to to blood) to approximately 500:1, and then 4 seconds after adding hemolysin, measure the absorbance of the sample mixture at a specific wavelength on the instrument's photometer, or manually introduce blood equipped with a photometer and an impedance measurement device In the analyzer, the hemoglobin concentration is measured or the white blood cell count is performed simultaneously.

More preferably, the leukocyte subsets are classified according to the volume size histogram obtained when the leukocyte count is performed.

In the most preferred manner, leukocytes can be divided into three subpopulations including lymphocytes, intermediate cells and granulocytes, wherein the intermediate cells include monocytes eosinophils and basophils.

By adopting the above scheme, the hemolysin that does not contain cyanide and is not toxic can be used to measure hemoglobin in a reliable way, and at the same time, white blood cells can be divided into at least two subgroups for separate counting.

Description of drawings

Fig. 1 is a spectrogram of the hemolysin of the present invention.

Fig. 2 is the white blood cell histogram obtained by the hemolysin of the present invention on the BC-3000+ hematology analyzer produced by Mindray Company.

Fig. 3 is an ultraviolet-visible absorption spectrum diagram of the hemolysin of the present invention when it is used as a diluent.

Fig. 4 is a white blood cell histogram obtained by the hemolysin of Example 2 of the present invention on the BC-3000+ blood analyzer produced by Mindray Company.

Detailed ways

In general, to measure the total hemoglobin concentration in a blood sample photometrically, a hemolytic reagent must be used to lyse the red blood cells and release the hemoglobin, which is then converted to a stable chromogen that can be detected at a predetermined wavelength white ultraviolet spectrum and determination. For quantitative and precise assays, the complexes formed need to be stable, at least over the time frame of the assay. Lysis of RBCs can be accomplished by acid lysis, osmolysis, and the use of various natural and synthetic surfactants. The released hemoglobin contains various forms, such as oxyhemoglobin, deoxyhemoglobin, methemoglobin, carboxyhemoglobin, etc. Most effective methods for converting hemoglobin into stable hemoglobin are to provide ligands that have a high affinity for heme iron. affinity to form stable hemoglobin complexes. This has been successfully demonstrated with the cyanide-hemoglobin method, in which the cyanide anion has an extremely high affinity for heme iron. The terms hemoglobin complex and hemoglobin chromogen are used interchangeably in the context of the present invention. Typically, the hemoglobin chromogen formed is not very stable in the absence of high affinity ligands. Its absorbance varies and in most cases decays with time. In this case, the analytical method is unreliable (even if the kinetics of the degradation reaction are well monitored and corrected because the chromogen may be very sensitive to the environment (e.g. temperature, sample preparation conditions, etc.), when a suitable hemoglobin ligand is provided , the hemoglobin transition can be quantified, and the stability of the formed hemoglobin complex ensures the reliability of the analytical method.

The choice of ligand depends on the analysis to be performed, eg, for hemoglobin determination only, or for multiple diagnostic assays (eg, hemoglobin determination combined with leukocyte count or leukocyte subpopulation count separately). A ligand that is excellent for hemoglobin determination may not be suitable for the latter application if the ligand is not compatible with the other assay. Sakata provides a method that can accurately measure hemoglobin in its literature. Anionic surfactants and nonionic surfactants are used to dissolve red blood cells to release hemoglobin, and the anionic surfactant SLS and hemoglobin in the reagent form SLS-hemoglobin complexes, There are peaks at 539nm and 572nm and have high time stability, but a big disadvantage is that white blood cells cannot be classified and counted at the same time, because white blood cells are also largely destroyed when non-ionic surfactants are used to dissolve red blood cells. up. Another disadvantage is that the time is very long, according to its literature, it takes 5-10 minutes, which is intolerable in the method of measuring with an instrument.

Generally speaking, anionic surfactants and cationic surfactants cannot be used together, because they will form anion-cation complexes, reduce solubility, form precipitation or emulsification in solution, and thus cannot be used. But in the present invention, through the appropriate ratio of the two, the complex formed will not form a precipitate, and the complex dissolves after adding a solubilizing agent. After the cationic surfactant dissolves red blood cells to release hemoglobin, due to the selected anionic surfactant The affinity with hemoglobin is much higher, and the anionic surfactant in the anion-cation surfactant complex is taken away to form a new surfactant-hemoglobin complex. By precisely controlling the concentration of the two, hemoglobin determination can be realized. On the other hand, the cationic surfactant destroys the leukocyte membrane, exposes the leukocyte nucleus, and performs size classification by a volumetric instrument.

The test also shows that the addition of nonionic surfactant and cationic surfactant greatly increases the speed of anionic surfactant-hemoglobin complex formation, and forms a stable complex within 3-5 seconds, making it possible to measure with an instrument. possible.

The cyanide-free hemolysin of the present invention is at least one surfactant selected from the group consisting of:

1) At least one cationic surfactant, which is capable of lysing all human red blood cells and dividing white blood cells into two subpopulations with a specific instrument within a suitable concentration range. Including quaternary ammonium salt, pyridinium salt, amine oxide quaternary ammonium salt.

R1 and R5 in the formula are long-chain alkyl, alkenyl or alkynyl groups with 8-18 carbon atoms, and R2, R3 and R4 are all short-chain alkyl, alkenyl, or alkynyl groups with 1-5 carbon atoms. group or hydrogen atom, x- is any kind of anion.

2) At least one anionic surfactant can combine with hemoglobin to form a chromogen at a suitable concentration, thereby measuring its concentration. The anionic surfactant has the following structure:

R ₁ -SO ₄ ^{-X +}

In the formula, R1 is a long-chain alkyl, alkenyl or alkynyl group with 8-18 carbon atoms, and x+ is any alkali metal cation or ammonium ion.

3) at least one nonionic surfactant having the following structure:

Wherein R1 is a long-chain alkyl group, alkenyl group or alkynyl group with 6-8 carbon atoms, and n is an integer of 4-40.

In a preferred form, the combination can be used to simultaneously provide a determination of total hemoglobin concentration, a count of leukocytes and a separate count of subpopulations of cells in a blood sample, wherein said leukocytes are differentiated to include lymphocytes, monocytes, and granulocytes. Three subpopulations of cells.

Among the cationic surfactants for lysing red blood cells, quaternary ammonium salts are preferred. The concentration of surfactant in the hemolysin composition needs to be sufficient to lyse red blood cells and release hemoglobin while preserving the white cell nuclei from destruction. In order to count leukocytes, it is not necessary to leave the leukocyte membrane intact. In general, when the hemolytic surfactant in the hemolysin composition of the present invention described above is used, the white blood cell membrane is partially lysed while the red blood cells are completely lysed and destroyed. The nuclei left by the leukocytes make it possible to count the leukocytes and separate the leukocyte subpopulations into lymphocytes, monocytes, and granulocytes using the direct current impedance method. The surfactant concentration in the hemolysin composition is about 2 g/L to about 100 g/L L, preferably about 15 g/L to about 60 g/L.

The concentration of anionic surfactant in hemolysin is sufficient to allow all hemoglobin to form an anionic surfactant-hemoglobin complex. If the amount of anionic surfactant is insufficient, the chromogen of the formed complex is destabilized. Tests have found that the concentration of the anionic surfactant in the present invention is in the range of 0.1-10g/L, preferably 0.5-3g/L, which is the most suitable.

In the present invention, the concentration range of the nonionic surfactant that we use depends on the concrete kind that adopts, but scope is in the scope of 0.5-5g/L, when it is proved through experiments that it is lower than 0.5g/L, it will not play a role. When it is higher than 5g/L, it will have a significant impact on the classification and determination of white blood cells, and even the determination of hemoglobin, and even cannot be classified.

The present invention also relates to a method for determining the total hemoglobin concentration in a blood sample, or simultaneously counting leukocytes or separately counting leukocyte subsets using the above-mentioned cyanide-free hemolysin composition.

Dilute the anticoagulated blood or peripheral blood with a suitable diluent, mix the appropriate amount of the above-mentioned hemolysin with the diluted sample (manually or automatically by the instrument), and the dilution ratio is from 125:1 (total reagent volume relative to to blood) to approximately 500:1, and then 4 seconds after adding hemolysin, measure the absorbance of the sample mixture at a specific wavelength on the instrument's photometer, or manually introduce blood equipped with a photometer and an impedance measurement device In the analyzer, the hemoglobin concentration is measured or the white blood cell count is performed at the same time, and more preferably, the white blood cell subsets are classified according to the volume size histogram obtained during the white blood cell count. In the most preferred manner, leukocytes can be divided into three subpopulations including lymphocytes, intermediate cells and granulocytes, wherein the intermediate cells include monocytes eosinophils and basophils.

Specifically, we use the BC-3000 blood analyzer produced by Mindray Company, take 13ul of venous anticoagulant blood, after dilution with 3.5ml of diluent, add 0.5ml of hemolysin according to the present invention, and perform white blood cell count and hemoglobin in the white blood cell channel Analysis, the histogram of the result is shown in Figure 2.

The hemolysin compositions and methods of using the compositions of the present invention provide accurate hemoglobin measurements, accurate white blood cell counts, and separate counts of white blood cell subsets. Table 1 shows that the hemolysin is highly correlated with the results of the five-category blood analyzer GENS in measuring the hemoglobin concentration using the BC-3000 blood analyzer produced by Mindray. There is an excellent correlation between the hemoglobin concentrations obtained on the BC-3000 and those obtained on the same instrument using classical hemolysin. Table 3 illustrates the excellent correlation between white blood cell counts obtained on the BC-3000 and those obtained on the same instrument using the instrument's companion hemolysin.

Table 1 Correlation coefficient between hemolysin results of the present invention and GENS results of Coulter Company. 1 2 3 4 5 average value Wbc 0.9983 0.9955 0.9971 0.9906 0.9799 0.9923 Hgb 0.9969 0.9935 0.9972 0.9981 0.9956 0.9963 Gran% 0.9650 0.9667 0.9543 0.9590 0.9281 0.9546 Lym% 0.9556 0.9585 0.9584 0.9428 0.9316 0.9494

The sample size for each trial was between 40-50.

The hemolysin compositions of the present invention provide accurate hemoglobin measurements in different patient samples. Among the samples we use, 70% are clinical patients from various departments, including infectious medicine, hematology, oncology, etc. Some of the blood samples from hematology patients contain different types of abnormal hemoglobin, but hemolysis of the present invention There is also a high degree of correlation between the element and method determination results and the commonly used cyanide-Hgb method (the correlation coefficient measured on the BC-3000 of Mindray Company is more than 0.99 and sees Table 1), which shows that the total hemoglobin concentration of various clinical samples is different. It can be determined by using the hemolysin composition of the present invention.

In general, the hemolysin compositions of the present invention comprising the above-described anionic surfactant ligands and surfactants can measure the total hemoglobin concentration of a blood sample over a very wide range of pH values. Preferably, the combination of one or several quaternary salts with any organic ligand is used to provide the determination of total hemoglobin concentration and white blood cell count in a blood sample by DC impedance assay methods. More preferably, the combination of one or more quaternary salts with an organic ligand compatible with differential analysis of leukocytes is used to provide determination of total hemoglobin concentration, leukocyte count and separate enumeration of leukocyte subpopulations of a blood sample. In the most preferred form, leukocytes are divided into three subpopulations including lymphocytes, monocytes and granulocytes.

The cyanide-free hemolysin composition and method using the composition have several advantages over prior art hemolysin assay methods. The invention makes it possible to accurately measure the concentration of hemoglobin in a blood sample under cyanide-free conditions, and to determine the number of white blood cells or to divide the white blood cell subgroups into lymphocytes, monocytes and granulocytes. The hemolysin composition rapidly converts hemoglobin present in a blood sample into a stable chromogen after about 5 seconds, allowing rapid automated analysis. Once formed, the hemoglobin complex is very stable during the assay period.

The hemolysin compositions of the invention can be used with a number of suitable blood thinners. We use formula cationic surfactant as dodecyltrimethylammonium bromide, its concentration is 25g/L, anionic surfactant is sodium lauryl sulfate (SLS), its concentration is 1.5g/L, non-ionic surface Active agent is the hemolysin of emulsifier OP, its concentration is 0.58g/L, uses standard phosphate buffered saline solution and commercial blood thinner to be produced by Mindray's M30 dilution, the whole blood sample processed by the above method, as shown in Figure 3 the graph shown. With these two diluents, essentially the same hemoglobin chromogen is formed, which can be measured at 540nm.

Unlike previous reagents, the hemolysin composition of the present invention has a wide range of pH values, and it was impossible for previous neutral and alkaline reagents to use this chemical substance for the determination of hemoglobin. Sakata in US Patent No. 5,242,832 states that if the pH value is 3.0 or lower, the destruction of leukocytes increases, thus making the measurement of leukocytes difficult. Fig. 2 shows a histogram of leukocyte subpopulation distribution obtained by using the hemolysin composition of the example (M30 as diluent) according to the method of the present invention. White blood cells are clearly divided into lymphocytes, monocytes and granulocytes.

Another way to use surfactants to stabilize the function of hemoglobin is to add it as a separate component to diluted blood, before or after adding cationic surfactants that can hemolyze, and the released hemoglobin can still interact with anions. Surfactants are determined by combining to form complexes.

If hemolysin is directly used to dissolve the blood sample without diluent, the concentration of each chemical component of hemolysin can be adjusted in a large range under a certain ratio, and it can be considered that adding a suitable electrolyte to achieve a suitable conductivity is suitable for a specific particle analysis instrument Used, if so used, the concentration range of the composition should be the same as the final concentration of hemolysin after complexing with the diluted blood sample.

Hemolysin may contain other optional additives, such as inorganic and organic salts, preservatives, antioxidants and all other chemical components that do not affect the determination of hemoglobin and white blood cell count or differential count.

Another way to use surfactants to stabilize the function of hemoglobin is to add it as a separate component to diluted blood, before or after adding cationic surfactants that can hemolyze, and the released hemoglobin can still interact with anions. Surfactants are determined by combining to form complexes.

Example 1

Prepare a solution in the following proportions:

1. Sodium lauryl sulfate 5g

2. Dodecyltrimethylammonium bromide 25g

3. Polyethylene glycol octylphenyl ether 5ml

4. Distilled water 1L

Use a G4 sand filter to circulate and filter, and use a suitable hematology analyzer or other particle analysis instruments to monitor the blank count value to meet the blank count requirements.

Pipette 13ul whole blood and add it to 3.5ml diluent, stir evenly, add 0.5ml hemolysin, and measure it on a BC series hematology analyzer.

Fig. 1 is the ultraviolet-visible absorption spectrum that is scanned immediately after the diluted blood is added with hemolysin. There is an absorption peak at 540nm.

Fig. 2 is the white blood cell histogram obtained by the hemolysin of the present invention on the BC-3000+ hematology analyzer produced by Mindray Company.

Fig. 3 is an ultraviolet-visible absorption spectrum diagram of the hemolysin of the present invention when it is used as a diluent.

Example 2

Prepare a solution in the following proportions:

1. Cetyltrimethylammonium chloride 10g

2. Sodium lauryl sulfate 1.5g

3. Emulsifier OP 0.58g

4. Double distilled water 1L

Use a G4 sand filter to circulate and filter, and use a suitable hematology analyzer or other particle analysis instruments to monitor the blank count value to meet the blank count requirements.

Dilute the blood sample with phosphate buffer saline (PBS) as a diluent, add the hemolysin of the above formula, measure the absorption UV-visible absorption spectrum as shown in Figure 3, and analyze it with a blood analyzer in the pre-dilution mode at the same time, the obtained The histogram is shown in Figure 4.

Claims (6)

1, a kind of cyanide-free hemolysin for comprising the aqueous solution of surfactant materials, is characterized in that the group of this surfactant for being made up of following material:

1) at least a cationic surfactant is selected from quaternary ammonium salt, pyridiniujm, amine oxide type quaternary ammonium salt,

Formula a formula b formula c

R1, R5 among its Chinese style a, formula b, the formula c is that carbon number is chain alkyl, the alkenyl or alkynyl of 8-18, and R2, R3, R4 are short-chain alkyl, thiazolinyl, alkynyl or the hydrogen atoms of carbon number 1-5 among formula a, the formula c, and x-is any negative ion;

2) at least a anionic surfactant, this anionic surfactant has following structure: R ₁-SO ₄ ^-X ⁺

R1 is that carbon number is chain alkyl, the alkenyl or alkynyl of 8-18 in the formula, x ⁺Be any alkali-metal kation or ammonium ion;

3) at least a non-ionic surfactant has following structure:

Wherein R1 is that carbon number is chain alkyl, the alkenyl or alkynyl of 6-8, and n is the integer of 4-40;

2, cyanide-free hemolysin according to claim 1 is characterized in that cationic surfactant concentration is 10g/L-25g/L.

3, cyanide-free hemolysin according to claim 1 is characterized in that anionic surfactant concentration is 1.5g/L-5g/L.

4, cyanide-free hemolysin according to claim 1 is characterized in that non-ionic surfactant concentration is 0.58g/L-5g/L.

5, cyanide-free hemolysin according to claim 1 is characterized in that cationic surfactant concentration is 25g/L, and the concentration of anionic surfactant is 1.5g/L, and the concentration of non-ionic surfactant is 0.58g/L.

6, cyanide-free hemolysin according to claim 1, the concentration that it is characterized in that the cationic surfactant DTAB is 25g/L, the concentration of anionic (SLS) is 1.5g/L, and the concentration of non-ionic surfactant polyoxyethylene nonylphenol ether is 0.58g/L.

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