CN111157713A - Hemolysin for blood analysis, preparation method thereof, reagent and kit - Google Patents
Hemolysin for blood analysis, preparation method thereof, reagent and kit Download PDFInfo
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- CN111157713A CN111157713A CN202010012477.7A CN202010012477A CN111157713A CN 111157713 A CN111157713 A CN 111157713A CN 202010012477 A CN202010012477 A CN 202010012477A CN 111157713 A CN111157713 A CN 111157713A
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- hemolysin
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- 108010006464 Hemolysin Proteins Proteins 0.000 title claims abstract description 53
- 239000003228 hemolysin Substances 0.000 title claims abstract description 53
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 37
- 238000004159 blood analysis Methods 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 239000004094 surface-active agent Substances 0.000 claims abstract description 33
- 239000006184 cosolvent Substances 0.000 claims abstract description 29
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 28
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims abstract description 26
- 239000012192 staining solution Substances 0.000 claims abstract description 16
- 239000000872 buffer Substances 0.000 claims abstract description 12
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims abstract 2
- 239000004606 Fillers/Extenders Substances 0.000 claims description 23
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 17
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- 239000012528 membrane Substances 0.000 claims description 15
- 239000006172 buffering agent Substances 0.000 claims description 14
- 239000011259 mixed solution Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- 239000008213 purified water Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 10
- GLDOVTGHNKAZLK-UHFFFAOYSA-N octadecan-1-ol Chemical group CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 claims description 10
- -1 pH regulator Substances 0.000 claims description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 8
- DDXLVDQZPFLQMZ-UHFFFAOYSA-M dodecyl(trimethyl)azanium;chloride Chemical group [Cl-].CCCCCCCCCCCC[N+](C)(C)C DDXLVDQZPFLQMZ-UHFFFAOYSA-M 0.000 claims description 7
- IWZKICVEHNUQTL-UHFFFAOYSA-M potassium hydrogen phthalate Chemical group [K+].OC(=O)C1=CC=CC=C1C([O-])=O IWZKICVEHNUQTL-UHFFFAOYSA-M 0.000 claims description 7
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims description 6
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 claims description 6
- 239000011976 maleic acid Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 235000002639 sodium chloride Nutrition 0.000 claims description 6
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 5
- GOQYKNQRPGWPLP-UHFFFAOYSA-N n-heptadecyl alcohol Natural products CCCCCCCCCCCCCCCCCO GOQYKNQRPGWPLP-UHFFFAOYSA-N 0.000 claims description 5
- 229940051841 polyoxyethylene ether Drugs 0.000 claims description 5
- 229920000056 polyoxyethylene ether Polymers 0.000 claims description 5
- 229960004063 propylene glycol Drugs 0.000 claims description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 5
- 235000011152 sodium sulphate Nutrition 0.000 claims description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 229920002675 Polyoxyl Polymers 0.000 claims description 3
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims description 3
- 229930182490 saponin Natural products 0.000 claims description 3
- 150000007949 saponins Chemical class 0.000 claims description 3
- 150000002148 esters Chemical class 0.000 claims description 2
- 239000003002 pH adjusting agent Substances 0.000 claims 1
- 239000013589 supplement Substances 0.000 claims 1
- 239000000975 dye Substances 0.000 abstract description 22
- 210000003743 erythrocyte Anatomy 0.000 abstract description 17
- 238000004043 dyeing Methods 0.000 abstract description 16
- 210000004369 blood Anatomy 0.000 abstract description 12
- 239000008280 blood Substances 0.000 abstract description 12
- 239000007788 liquid Substances 0.000 abstract description 7
- 238000004458 analytical method Methods 0.000 abstract description 6
- 210000003912 basophilic leucocyte Anatomy 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 17
- QGKMIGUHVLGJBR-UHFFFAOYSA-M (4z)-1-(3-methylbutyl)-4-[[1-(3-methylbutyl)quinolin-1-ium-4-yl]methylidene]quinoline;iodide Chemical compound [I-].C12=CC=CC=C2N(CCC(C)C)C=CC1=CC1=CC=[N+](CCC(C)C)C2=CC=CC=C12 QGKMIGUHVLGJBR-UHFFFAOYSA-M 0.000 description 15
- 210000003651 basophil Anatomy 0.000 description 14
- 238000001514 detection method Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- 210000000265 leukocyte Anatomy 0.000 description 7
- 230000002209 hydrophobic effect Effects 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 239000011148 porous material Substances 0.000 description 6
- 238000010186 staining Methods 0.000 description 5
- MZZINWWGSYUHGU-UHFFFAOYSA-J ToTo-1 Chemical compound [I-].[I-].[I-].[I-].C12=CC=CC=C2C(C=C2N(C3=CC=CC=C3S2)C)=CC=[N+]1CCC[N+](C)(C)CCC[N+](C)(C)CCC[N+](C1=CC=CC=C11)=CC=C1C=C1N(C)C2=CC=CC=C2S1 MZZINWWGSYUHGU-UHFFFAOYSA-J 0.000 description 3
- GRRMZXFOOGQMFA-UHFFFAOYSA-J YoYo-1 Chemical compound [I-].[I-].[I-].[I-].C12=CC=CC=C2C(C=C2N(C3=CC=CC=C3O2)C)=CC=[N+]1CCC[N+](C)(C)CCC[N+](C)(C)CCC[N+](C1=CC=CC=C11)=CC=C1C=C1N(C)C2=CC=CC=C2O1 GRRMZXFOOGQMFA-UHFFFAOYSA-J 0.000 description 3
- 238000000149 argon plasma sintering Methods 0.000 description 3
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- 238000010586 diagram Methods 0.000 description 3
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- 238000002835 absorbance Methods 0.000 description 2
- IOJUPLGTWVMSFF-UHFFFAOYSA-N benzothiazole Chemical compound C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 description 2
- 210000001772 blood platelet Anatomy 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
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- 125000000355 1,3-benzoxazolyl group Chemical group O1C(=NC2=C1C=CC=C2)* 0.000 description 1
- INGWEZCOABYORO-UHFFFAOYSA-N 2-(furan-2-yl)-7-methyl-1h-1,8-naphthyridin-4-one Chemical compound N=1C2=NC(C)=CC=C2C(O)=CC=1C1=CC=CO1 INGWEZCOABYORO-UHFFFAOYSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- ULHRKLSNHXXJLO-UHFFFAOYSA-L Yo-Pro-1 Chemical compound [I-].[I-].C1=CC=C2C(C=C3N(C4=CC=CC=C4O3)C)=CC=[N+](CCC[N+](C)(C)C)C2=C1 ULHRKLSNHXXJLO-UHFFFAOYSA-L 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
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- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000008676 import Effects 0.000 description 1
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- 230000000877 morphologic effect Effects 0.000 description 1
- 238000000569 multi-angle light scattering Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
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- ACOJCCLIDPZYJC-UHFFFAOYSA-M thiazole orange Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1.C1=CC=C2C(C=C3N(C4=CC=CC=C4S3)C)=CC=[N+](C)C2=C1 ACOJCCLIDPZYJC-UHFFFAOYSA-M 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N2021/1734—Sequential different kinds of measurements; Combining two or more methods
Abstract
The invention discloses hemolysin for blood analysis, a preparation method, a reagent and a kit thereof, belonging to the technical field of blood analysis. The hemolysin for blood analysis comprises the following components: quaternary ammonium salt surfactant, nonionic surfactant, buffer, pH regulator, and conductivity replenisher. The reagent for blood analysis comprises a staining solution and the hemolysin; the dyeing liquid comprises the following components: asymmetric cyanine dyes and cosolvents. The reagent provided by the embodiment of the invention only consists of hemolysin and a staining solution, and can replace an imported original reagent to realize that the nucleated red blood cells and basophilic leukocytes in blood are simultaneously and accurately detected in one channel, so that the blood analysis cost can be reduced, and the convenience of analysis operation can be improved.
Description
Technical Field
The invention relates to the technical field of blood analysis, in particular to hemolysin for blood analysis, a preparation method thereof, a reagent and a kit.
Background
Due to clinical needs, there is an increasing demand for five-class blood analyzers. Based on different classification principles of leucocytes and nucleated erythrocytes of various manufacturers, the used reagents are different. The classification principle of the mainstream five-classification blood analyzer mainly comprises a high-frequency conductance laser scattering combined detection method (namely VCS technology), a light scattering and cell staining combined detection method, an electrical impedance and radio frequency conductance combined detection method, a multi-angle laser polarization light scattering detection method and the like, but the method is not limited to an optical method and staining technology, and used reagents mainly comprise hemolysin and staining solution. However, most five-classification hemoglobins detect nucleated red blood cells and basophils through two channels, namely, the nucleated red blood cells require one hemolysin, the basophils require another different hemolysin, and the operation is inconvenient.
In addition, although reagents for detecting basophils and nucleated red blood cells are available in the prior art, most of the reagents are imported from abroad, namely, the existing nucleated red blood cells and basophils analysis reagents have the problems of high reagent formula difficulty, high use cost, long-term dependence on import, complicated purchase channels and the like, and cannot meet the normal use of domestic hospitals in time. Therefore, it is necessary to develop a blood analysis reagent which has a simple formula and a low cost, can replace imported original reagents, and can reduce the dependence on foreign imported products.
Disclosure of Invention
An object of an embodiment of the present invention is to provide hemolysin for blood analysis to solve the problems set forth in the background art described above.
In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:
a hemolysin for blood analysis, said hemolysin comprising per liter the following components: 0.1-2 g of quaternary ammonium salt surfactant, 1-5 g of nonionic surfactant, 5-10 g of buffering agent, 1-5 g of pH regulator and 1-5 g of conductivity extender.
As a preferable mode of the embodiment of the present invention, the hemolysin includes the following components per liter: 0.5-1.5 g of quaternary ammonium salt surfactant, 2.5-4 g of nonionic surfactant, 6-8 g of buffering agent, 2-3 g of pH regulator and 3-4 g of conductivity extender; the quaternary ammonium salt surfactant is dodecyl trimethyl ammonium chloride; the nonionic surfactant is a polyoxyl ester surfactant or a saponin surfactant; the buffer is potassium hydrogen phthalate.
In another preferred embodiment of the present invention, the nonionic surfactant is stearyl alcohol polyoxyethylene ether.
As another preferable scheme of the embodiment of the invention, the pH regulator is one or more of formic acid, citric acid and maleic acid. Preferably, the pH regulator is maleic acid.
As another preferred embodiment of the present invention, the conductivity extender is one or more of potassium chloride, sodium chloride and sodium sulfate. Preferably, the conductivity extender is sodium chloride.
Another object of an embodiment of the present invention is to provide a method for preparing hemolysin, which comprises the following steps:
weighing quaternary ammonium salt surfactant, nonionic surfactant, buffer, pH regulator and conductivity replenisher according to the content of each liter of the components of the hemolysin for later use;
adding the weighed quaternary ammonium salt surfactant, nonionic surfactant, pH regulator, buffer and conductivity extender into purified water in sequence, stirring and mixing, and continuously adding purified water to perform constant volume to obtain a mixed solution;
and filtering the mixed solution by using a microporous filter membrane with the aperture of 0.1-0.3 mu m to obtain the hemolysin.
The other purpose of the embodiment of the invention is to provide hemolysin prepared by the preparation method.
Another object of an embodiment of the present invention is to provide a reagent for blood analysis, which includes a staining solution and the hemolysin.
As another preferable scheme of the embodiment of the invention, each liter of the dyeing solution comprises the following components: 0.1-0.5 g of asymmetric cyanine dye, and the balance of cosolvent; the cosolvent is one or more of ethylene glycol, methanol and 1, 2-propylene glycol. Preferably, the cosolvent is ethylene glycol.
As another preferable embodiment of the present invention, the preparation method of the dyeing solution comprises the following steps:
weighing asymmetric cyanine dyes and cosolvents according to the content of components contained in each liter of the dyeing liquid for later use;
and adding the weighed asymmetric cyanine dyes into a cosolvent, stirring and mixing, carrying out constant volume by using the cosolvent, and filtering by using a hydrophobic microporous filter membrane with the pore diameter of 0.1-0.3 mu m to obtain the dyeing solution.
It is another object of the embodiments of the present invention to provide a kit for blood analysis, which contains the above-mentioned reagents.
Compared with the prior art, the embodiment of the invention has the beneficial effects that:
the hemolysin provided by the embodiment of the invention has the advantages of easily obtained raw materials and simple preparation method, and can be matched with a staining solution to accurately analyze and detect nucleated red blood cells and basophils in blood, so that the blood analysis cost can be reduced and the convenience of analysis operation can be improved. In addition, the reagent provided by the embodiment of the invention only consists of hemolysin and a staining solution, and can replace an imported original reagent to realize the simultaneous accurate detection of the nucleated red blood cells and the basophilic leukocytes in the blood in one channel; the hemolysin can dissolve the modified cells, the staining solution stains nucleic acid of nucleated red blood cells and basophils, and the two types of cells can be detected by multi-angle light scattering. Compared with the traditional two-channel detection technology, the technical scheme of the invention has the advantages that: one reagent is reduced, basophils are also detected by a staining method, and the counting is more accurate.
In particular, the reagent is suitable for a blood analyzer based on a nucleic acid staining agent principle, such as a Hessemcon XN series five-classification blood analyzer. The technical principle is that a whole blood sample and hemolysin are fully and uniformly mixed in an WNR detection pool of an instrument, a staining solution is added for incubation, the hemolysin modifies and fixes the shapes of basophil and nucleated red blood cells in the process so that the basophil and the nucleated red blood cells keep natural states, the nucleated red blood cells and the basophil are stained in different degrees, and membranes (cell membranes, nuclear membranes and particle membranes) of the cells are also stained in different degrees. The nucleated red blood cells and basophils have different staining degrees on staining fluid, and the specific nuclear morphology and particle structure of each cell cause different light scattering intensities, so that specific absorbance and scattered light intensities are generated. Therefore, nucleated erythrocytes and basophils can be detected in the scattergram according to the difference in absorbance and scattered light intensity.
Drawings
FIG. 1 is a standard scatter plot of blood analysis.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
This embodiment provides a reagent for blood analysis, which includes hemolysin and a staining solution. The preparation method of the hemolysin comprises the following steps:
s11, weighing 0.1g of quaternary ammonium salt surfactant, 1g of nonionic surfactant, 5g of buffering agent, 1g of pH regulator and 1g of conductivity extender for later use; wherein the quaternary ammonium salt surfactant is dodecyl trimethyl ammonium chloride; the nonionic surfactant is a commercially available polyoxyl alcohol ester surfactant; the buffering agent is potassium hydrogen phthalate; the pH regulator is formic acid; the conductivity extender is potassium chloride.
S12, sequentially adding the weighed quaternary ammonium salt surfactant, nonionic surfactant, pH regulator, buffer and conductivity extender into 600mL of purified water, stirring, dissolving and mixing, and continuously adding purified water to a constant volume of 1L to obtain a mixed solution.
S13, filtering the mixed solution by using a microporous filter membrane with the aperture of 0.1 mu m to obtain the hemolysin.
In addition, the preparation method of the dyeing liquid comprises the following steps:
s21, weighing 0.1g of asymmetric cyanine dye and 1L of cosolvent for later use. Wherein the cosolvent is methanol.
S22, adding the weighed asymmetric cyanine dyes into 600mL of cosolvent, stirring, dissolving and mixing, continuing to perform constant volume to 1L with the cosolvent, and then filtering with a hydrophobic microporous filter membrane with the pore diameter of 0.1 mu m to obtain the dyeing solution.
Example 2
This embodiment provides a reagent for blood analysis, which includes hemolysin and a staining solution. The preparation method of the hemolysin comprises the following steps:
s11, weighing 2g of quaternary ammonium salt surfactant, 5g of nonionic surfactant, 10g of buffering agent, 5g of pH regulator and 5g of conductivity extender for later use; wherein the quaternary ammonium salt surfactant is dodecyl trimethyl ammonium chloride; the nonionic surfactant is commercially available saponin surfactant; the buffering agent is potassium hydrogen phthalate; the pH regulator is a mixture of formic acid, citric acid and maleic acid which are mixed according to equal mass ratio; the conductivity extender is a mixture of potassium chloride, sodium chloride and sodium sulfate which are mixed according to equal mass ratio.
S12, sequentially adding the weighed quaternary ammonium salt surfactant, nonionic surfactant, pH regulator, buffer and conductivity extender into 600mL of purified water, stirring, dissolving and mixing, and continuously adding purified water to a constant volume of 1L to obtain a mixed solution.
S13, filtering the mixed solution by using a microporous filter membrane with the aperture of 0.3 mu m to obtain the hemolysin.
In addition, the preparation method of the dyeing liquid comprises the following steps:
s21, weighing 0.5g of asymmetric cyanine dye and 1L of cosolvent for later use. Wherein, the cosolvent is a mixture of glycol, methanol and 1, 2-propylene glycol mixed according to equal volume.
S22, adding the weighed asymmetric cyanine dyes into 600mL of cosolvent, stirring, dissolving and mixing, continuing to perform constant volume to 1L with the cosolvent, and then filtering with a hydrophobic microporous filter membrane with the pore diameter of 0.3 mu m to obtain the dyeing solution.
Example 3
This embodiment provides a reagent for blood analysis, which includes hemolysin and a staining solution. The preparation method of the hemolysin comprises the following steps:
s11, weighing 0.5g of quaternary ammonium salt surfactant, 2.5g of nonionic surfactant, 6g of buffering agent, 2g of pH regulator and 3g of conductivity extender for later use; wherein the quaternary ammonium salt surfactant is dodecyl trimethyl ammonium chloride; the nonionic surfactant is stearyl alcohol polyoxyethylene ether; the buffering agent is potassium hydrogen phthalate; the pH regulator is citric acid; the conductivity extender is sodium sulfate.
S12, sequentially adding the weighed quaternary ammonium salt surfactant, nonionic surfactant, pH regulator, buffer and conductivity extender into 600mL of purified water, stirring, dissolving and mixing, and continuously adding purified water to a constant volume of 1L to obtain a mixed solution.
S13, filtering the mixed solution by using a microporous filter membrane with the aperture of 0.2 mu m to obtain the hemolysin.
In addition, the preparation method of the dyeing liquid comprises the following steps:
s21, weighing 0.3g of asymmetric cyanine dye and 1L of cosolvent for later use. Wherein the cosolvent is 1, 2-propylene glycol.
S22, adding the weighed asymmetric cyanine dyes into 600mL of cosolvent, stirring, dissolving and mixing, continuing to perform constant volume to 1L with the cosolvent, and then filtering with a hydrophobic microporous filter membrane with the pore diameter of 0.2 mu m to obtain the dyeing solution.
Example 4
This embodiment provides a reagent for blood analysis, which includes hemolysin and a staining solution. The preparation method of the hemolysin comprises the following steps:
s11, weighing 1.5g of quaternary ammonium salt surfactant, 4g of nonionic surfactant, 8g of buffering agent, 3g of pH regulator and 4g of conductivity extender for later use; wherein the quaternary ammonium salt surfactant is dodecyl trimethyl ammonium chloride; the nonionic surfactant is stearyl alcohol polyoxyethylene ether; the buffering agent is potassium hydrogen phthalate; the pH regulator is a mixture of citric acid and maleic acid in equal mass ratio; the conductivity extender is a mixture of sodium chloride and sodium sulfate in equal mass ratio.
S12, sequentially adding the weighed quaternary ammonium salt surfactant, nonionic surfactant, pH regulator, buffer and conductivity extender into 600mL of purified water, stirring, dissolving and mixing, and continuously adding purified water to a constant volume of 1L to obtain a mixed solution.
S13, filtering the mixed solution by using a microporous filter membrane with the aperture of 0.2 mu m to obtain the hemolysin.
In addition, the preparation method of the dyeing liquid comprises the following steps:
s21, weighing 0.3g of asymmetric cyanine dye and 1L of cosolvent for later use. Wherein, the cosolvent is a mixture of ethylene glycol and 1, 2-propylene glycol in equal volume ratio.
S22, adding the weighed asymmetric cyanine dyes into 600mL of cosolvent, stirring, dissolving and mixing, continuing to perform constant volume to 1L with the cosolvent, and then filtering with a hydrophobic microporous filter membrane with the pore diameter of 0.2 mu m to obtain the dyeing solution.
Example 5
This embodiment provides a reagent for blood analysis, which includes hemolysin and a staining solution. The preparation method of the hemolysin comprises the following steps:
s11, weighing 1g of quaternary ammonium salt surfactant, 3g of nonionic surfactant, 7g of buffering agent, 2.5g of pH regulator and 3.5g of conductivity extender for later use; wherein the quaternary ammonium salt surfactant is dodecyl trimethyl ammonium chloride; the nonionic surfactant is stearyl alcohol polyoxyethylene ether; the buffering agent is potassium hydrogen phthalate; the pH regulator is maleic acid; the conductivity extender is sodium chloride.
S12, sequentially adding the weighed quaternary ammonium salt surfactant, nonionic surfactant, pH regulator, buffer and conductivity extender into 600mL of purified water, stirring, dissolving and mixing, and continuously adding purified water to a constant volume of 1L to obtain a mixed solution.
S13, filtering the mixed solution by using a microporous filter membrane with the aperture of 0.2 mu m to obtain the hemolysin.
In addition, the preparation method of the dyeing liquid comprises the following steps:
s21, weighing 0.3g of asymmetric cyanine dye and 1L of cosolvent for later use. Wherein the cosolvent is ethylene glycol.
S22, adding the weighed asymmetric cyanine dyes into 600mL of cosolvent, stirring, dissolving and mixing, continuing to perform constant volume to 1L with the cosolvent, and then filtering with a hydrophobic microporous filter membrane with the pore diameter of 0.2 mu m to obtain the dyeing solution.
It should be noted that the asymmetric cyanine dyes described in examples 1 to 5 can be a mixture of the commercially available TOTO dye and YOYO dye in an equal mass ratio. Among them, TOTO and YOYO are a class of asymmetric cyanine dyes developed by Glazer et al that have high affinity for nucleic acids. The dye has no fluorescence in the solution, so that the fluorescence background interference in the detection process is reduced, and the dye can generate strong fluorescence after being combined with nucleic acid; they are connected as dimers from the monomers thiazole orange TO and oxazole yellow YO, respectively, via a flexible chain of polymethylene amines. YOYO is an analog of TOTO except that the benzothiazole is replaced with benzoxazole, which has the formula:
comparative example 1
This comparative example provides an imported original reagent for blood analysis, which is a commercially available product of Sysmex-XN (B4) of Sysmex.
Under the same experimental environment, 30 blood samples were taken, and the detection and analysis were performed by using the reagent provided in example 5 and the reagent provided in comparative example 1, respectively, and the analysis results are shown in tables 1 to 2 below. Wherein, the blood analysis instrument adopts a five-classification blood analysis instrument of the Xisenmeikang XN series, and the detection channel is WNR. The detection principle is as follows: mixing the blood specimen and hemolysin uniformly, reacting in an incubation cup, wherein the surfactant can dissolve red blood cells and platelets, the nucleated red blood cells are dissolved into naked nuclei, the white blood cells except basophils keep the original shape, other cells such as lymph, mononuclear cells, neutral white blood cells and the like are dissolved into naked nuclei, and the volume of the nucleated red blood cells is far smaller than that of the white blood cells; then adding a dye, and dyeing the nucleic acid substances in the cells by the dye; the sheath fluid without particle particles surrounds the sample by hydraulic action, each cell passes through the central axis of the flow cell in a single longitudinal column form and is irradiated by a laser beam, the instrument can convert signals into electric signals due to different degrees of fluorescence intensity (SFL) and scattered light intensity (FSC) of each particle, the quantity and morphological characteristics of each tangible component are obtained by a scattering diagram (as shown in figure 1, and figure 1 is a standard scattering diagram), Nucleated Red Blood Cells (NRBC), basophilic white blood cells (BA) and other White Blood Cells (WBC) are classified and counted, and various cells are displayed in a three-dimensional scattering diagram; in addition, the cells are irradiated by laser with the wavelength of 633nm, and the obtained forward scattered light (FSC), side scattered light (SSC) and Side Fluorescence (SFL) can count and classify the cells in the blood sample; wherein, FSC and SSC reflect cell surface structure, particle shape, nuclear form, refractive index, reflectivity and the like, generally, the larger the cell is, the stronger the signal of FSC is, and the more complicated the cell internal structure is, the stronger the signal of SSC is; SFL mainly reflects the kind and abundance of nucleic acids and small organs. These 3 kinds of signals can be used to classify and count leukocytes, nucleated erythrocytes, reticulocytes, and platelets, and can also be used to detect abnormal cells and immature cells, using conventional digital techniques and algorithms.
TABLE 1
TABLE 2
As can be seen from tables 1-2 above, the reagent provided in the embodiment of the present invention, which only contains one hemolysin and one staining solution, can simultaneously and accurately detect the content of nucleated red blood cells and basophils in blood, has a detection and analysis effect similar to that of the existing commercially available imported original reagent, and can be used in place of the original reagent, so as to reduce the research and development cost of blood analysis and the reagent use cost of each medical institution, and solve the problem of dependence of each medical institution on the imported reagent.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (10)
1. A hemolysin for blood analysis, said hemolysin comprising per liter the following components: 0.1-2 g of quaternary ammonium salt surfactant, 1-5 g of nonionic surfactant, 5-10 g of buffering agent, 1-5 g of pH regulator and 1-5 g of conductivity extender.
2. A hemolysin for blood analysis according to claim 1, characterized in that said hemolysin comprises the following composition per liter: 0.5-1.5 g of quaternary ammonium salt surfactant, 2.5-4 g of nonionic surfactant, 6-8 g of buffering agent, 2-3 g of pH regulator and 3-4 g of conductivity extender; the quaternary ammonium salt surfactant is dodecyl trimethyl ammonium chloride; the nonionic surfactant is a polyoxyl ester surfactant or a saponin surfactant; the buffer is potassium hydrogen phthalate.
3. A hemolysin according to claim 2, characterized in that said non-ionic surfactant is stearyl alcohol polyoxyethylene ether.
4. A hemolysin according to claim 1 or 2, wherein said pH adjusting agent is one or more of formic acid, citric acid and maleic acid.
5. A hemolysin according to claim 1 or 2, characterized in that said conductivity supplement is one or more of potassium chloride, sodium chloride and sodium sulfate.
6. A method for the preparation of a hemolysin according to any one of claims 1 to 5, comprising the steps of:
weighing quaternary ammonium salt surfactant, nonionic surfactant, buffer, pH regulator and conductivity replenisher according to the content of each liter of the components of the hemolysin for later use;
adding the weighed quaternary ammonium salt surfactant, nonionic surfactant, pH regulator, buffer and conductivity extender into purified water in sequence, stirring and mixing, and continuously adding purified water to perform constant volume to obtain a mixed solution;
and filtering the mixed solution by using a microporous filter membrane with the aperture of 0.1-0.3 mu m to obtain the hemolysin.
7. A hemolysin produced by the method of claim 6.
8. A reagent for blood analysis comprising a staining solution, characterized by further comprising the hemolysin according to any one of claims 1 to 5 and claim 7.
9. The reagent for blood analysis according to claim 8, wherein the staining solution comprises the following components per liter: 0.1-0.5 g of asymmetric cyanine dye, and the balance of cosolvent; the cosolvent is one or more of ethylene glycol, methanol and 1, 2-propylene glycol.
10. A kit for blood analysis, comprising the reagent according to claim 8 or 9.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111781047A (en) * | 2020-07-03 | 2020-10-16 | 四川恒健生物科技有限公司 | Hemolysin for blood analysis and preparation method and reagent thereof |
| CN113351062A (en) * | 2021-07-14 | 2021-09-07 | 上海可恩口腔门诊部有限公司 | Formula and manufacturing device of hemocyte analyzer hemolysin reagent |
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