CN112285337A - Sheath fluid for animal hemocyte analyzer and preparation method thereof - Google Patents
Sheath fluid for animal hemocyte analyzer and preparation method thereof Download PDFInfo
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- CN112285337A CN112285337A CN202011101309.1A CN202011101309A CN112285337A CN 112285337 A CN112285337 A CN 112285337A CN 202011101309 A CN202011101309 A CN 202011101309A CN 112285337 A CN112285337 A CN 112285337A
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- 239000012530 fluid Substances 0.000 title claims abstract description 74
- 241001465754 Metazoa Species 0.000 title claims abstract description 26
- 210000003677 hemocyte Anatomy 0.000 title claims abstract description 14
- 229940000351 hemocyte Drugs 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 239000002518 antifoaming agent Substances 0.000 claims abstract description 52
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- 239000007788 liquid Substances 0.000 claims abstract description 34
- 239000007853 buffer solution Substances 0.000 claims abstract description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 230000003204 osmotic effect Effects 0.000 claims abstract description 14
- 239000000022 bacteriostatic agent Substances 0.000 claims abstract description 13
- XMGKYXFDYYTZFD-UHFFFAOYSA-N 4-amino-1-hydroxy-2-methylbutane-2-sulfonic acid Chemical compound OCC(C)(S(O)(=O)=O)CCN XMGKYXFDYYTZFD-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 14
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- 239000011780 sodium chloride Substances 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 9
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- YNLCVAQJIKOXER-UHFFFAOYSA-N N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid Chemical compound OCC(CO)(CO)NCCCS(O)(=O)=O YNLCVAQJIKOXER-UHFFFAOYSA-N 0.000 abstract description 14
- 238000012360 testing method Methods 0.000 abstract description 12
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- 238000005187 foaming Methods 0.000 abstract description 3
- 239000008055 phosphate buffer solution Substances 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 description 28
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical group Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 24
- 210000000265 leukocyte Anatomy 0.000 description 20
- 210000004369 blood Anatomy 0.000 description 18
- 239000008280 blood Substances 0.000 description 18
- 230000000694 effects Effects 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- 239000006172 buffering agent Substances 0.000 description 10
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- 241000894007 species Species 0.000 description 4
- LUQKBGZRUIKUJG-UHFFFAOYSA-N 3-(trihydroxymethylamino)propane-1-sulfonic acid Chemical compound OC(O)(O)NCCCS(O)(=O)=O LUQKBGZRUIKUJG-UHFFFAOYSA-N 0.000 description 3
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
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- 240000005578 Rivina humilis Species 0.000 description 3
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- 239000008213 purified water Substances 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 description 3
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 210000003651 basophil Anatomy 0.000 description 2
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- 244000144993 groups of animals Species 0.000 description 2
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- 230000005764 inhibitory process Effects 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- MCSINKKTEDDPNK-UHFFFAOYSA-N propyl propionate Chemical compound CCCOC(=O)CC MCSINKKTEDDPNK-UHFFFAOYSA-N 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- QARJWQSAAYUDJA-UHFFFAOYSA-N 3-(aminomethyl)-1,4-dihydroxy-3-(hydroxymethyl)-2-methylbutane-2-sulfonic acid Chemical compound OCC(C)(S(O)(=O)=O)C(CN)(CO)CO QARJWQSAAYUDJA-UHFFFAOYSA-N 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
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- 241001494479 Pecora Species 0.000 description 1
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- 238000002835 absorbance Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
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- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 239000006177 biological buffer Substances 0.000 description 1
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- 238000004159 blood analysis Methods 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- VCMFGDACKBCWDX-UHFFFAOYSA-K dipotassium sodium hydrogen phosphate hydroxide Chemical compound [OH-].[Na+].P(=O)(O)([O-])[O-].[K+].[K+] VCMFGDACKBCWDX-UHFFFAOYSA-K 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- BSOPSBIGIYEHGT-UHFFFAOYSA-N dodecyl 2-phenylacetate Chemical compound CCCCCCCCCCCCOC(=O)CC1=CC=CC=C1 BSOPSBIGIYEHGT-UHFFFAOYSA-N 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
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- 239000003999 initiator Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 229960005323 phenoxyethanol Drugs 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
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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
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
- G01N33/491—Blood by separating the blood components
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Ecology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a sheath fluid for a veterinary hemocyte analyzer, which comprises the following components: the antibacterial agent comprises a buffer solution, a bacteriostatic agent, a defoaming agent, an osmotic pressure regulator, a pH value regulator and pure water, wherein the buffer solution comprises N-tri (hydroxymethyl) methyl-3-aminopropanesulfonic acid; the pH value range of the sheath liquid is controlled to be 7.8-8.8. Compared with the traditional phosphate buffer solution or borate buffer solution, the N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid can better maintain the cell morphology, the invention also provides a preparation method and a storage mode of the sheath liquid, a deoxidizer is placed on the packaging cover during storage, the addition of the defoamer and the use of the deoxidizer both reduce the foaming degree of the sheath liquid and improve the test precision of the blood cells of the animals.
Description
Technical Field
The invention relates to a sheath fluid and a preparation method thereof, in particular to a sheath fluid for a veterinary hemocyte analyzer and a preparation method thereof.
Background
Blood cell analysis is the most common item in clinical examination, adopts a blood cell analysis technology, can monitor the physical condition in time by observing the change of the number of blood cells and the morphological distribution, has great help for screening blood diseases, treating after healing and the like, and is one of the common auxiliary examination means for doctors to diagnose the disease condition. At present, the analysis technology of human blood cells in China is mature, and especially, the white blood cells are developed from three categories to five categories, so that the automation is completely realized. However, automated analysis of veterinary blood cells has been less studied.
The morphological structure and size of blood cells vary greatly between human and veterinary species, and also between species of veterinary species. In the blood cell analysis, a detected sample is wrapped by sheath fluid and passes through a detection part, and is irradiated by laser to identify or count a cell structure, so that the cleanliness, the absorbance, the pH value and the osmotic pressure of the sheath fluid all meet related requirements, otherwise, the detection result of an instrument is influenced, and therefore, a reagent more suitable for the morphological analysis of the blood cells of the beasts is used when the blood cell analyzer is used for classifying the white blood cells. At present, in many cases, a human blood cell classification reagent is directly applied to a veterinary blood cell analyzer, certain irrationality exists, and great influence is generated on the analysis precision of the veterinary blood cells.
Disclosure of Invention
The invention aims to provide a sheath fluid special for a veterinary hematology analyzer, improve the accuracy of veterinary blood cell analysis and provide the sheath fluid for the veterinary blood cell analyzer.
In order to achieve the above object, the present application provides a sheath fluid for a veterinary hemocyte analyzer, comprising the following components: the antibacterial agent comprises a buffer solution, a bacteriostatic agent, a defoaming agent, an osmotic pressure regulator, a pH value regulator and pure water, wherein the buffer solution comprises N-tri (hydroxymethyl) methyl-3-aminopropanesulfonic acid; the pH value range of the sheath liquid is controlled to be 7.8-8.8.
As a further refinement of the present application, the buffer solution further comprises Tris.
As a further improvement of the application, the defoaming agent is a GPES type defoaming agent, the bacteriostatic agent is ethylene glycol phenyl ether, and the osmotic pressure regulator is inorganic salt.
As a further improvement of the present application, the mass concentration of each of the constituent components in the sheath fluid is as follows: the buffer solution is 0.5-1.0 g/L, the ethylene glycol phenyl ether is 3-8 g/L, and the GPES defoaming agent is 0.001-0.05 g/L.
As a further refinement of the present application, the inorganic salt is sodium chloride.
As a further improvement of the present application, in 1L of the sheath fluid, 5.5g of the ethylene glycol phenyl ether; the Tris0.25g; 0.55g of N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid; 0.002 g-0.01 g of GPES defoaming agent; 11g of the sodium chloride; the balance of pH value regulator and pure water; the pH value of the sheath liquid is 7.8-8.8.
In order to achieve the above object, the present application also provides a method for preparing a sheath fluid for a veterinary hemocyte analyzer, comprising the following steps: s1, sequentially dissolving the buffer solution, the bacteriostatic agent, the defoaming agent and the osmotic pressure regulator into pure water, and performing constant volume to obtain a mixed solution; s2, adjusting the pH value of the mixed solution in the step S1 by using a pH value regulator, and controlling the pH value range of the mixed solution to be 7.8-8.8 to obtain a sheath liquid for the animal hemocyte analyzer; s3, filtering the sheath liquid in the step S2 by adopting a 0.22 mu m polypropylene membrane, and storing the sheath liquid in a sealed way by using a container.
As a further improvement of the present application, the container includes a closure cap, and a deoxidizer is placed on the closure cap.
As a further improvement of the present application, the deoxidizer is an organic matrix deoxidizer.
As a further improvement of the application, the concentration of the organic matrix deoxidizer is 0.01 g/L.
The sheath fluid for the veterinary hemocyte analyzer has the beneficial effects that the sheath fluid comprises the following components: the antibacterial agent comprises a buffer solution, a bacteriostatic agent, a defoaming agent, an osmotic pressure regulator, a pH value regulator and pure water, wherein the buffer solution comprises N-tri (hydroxymethyl) methyl-3-aminopropanesulfonic acid; the pH value range of the sheath liquid is controlled to be 7.8-8.8. Compared with the traditional phosphate or borate buffer solution, the N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid can better maintain the cell morphology, the addition of the defoaming agent greatly reduces the foaming degree of the sheath solution, and the test precision of the blood cells of the animals is improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the specific embodiments of the present application. It should be apparent that the described embodiments are only some of the embodiments of the present application, 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 application.
In order to improve the accuracy of analyzing the blood cells of the animals, the invention provides a sheath fluid for a blood cell analyzer for the animals, which comprises the following components: the antibacterial agent comprises a buffer solution, a bacteriostatic agent, a defoaming agent, an osmotic pressure regulator, a pH value regulator and pure water, wherein the buffer solution comprises N-tri (hydroxymethyl) methyl-3-aminopropanesulfonic acid. The pH value regulator is mainly used for regulating the pH value of the sheath liquid, the pH value range of the sheath liquid is controlled to be 7.8-8.8, and the pH value regulator can be hydrochloric acid and the like. N-tri (hydroxymethyl) methyl-3-aminopropanesulfonic acid is one of biological buffering agents, and compared with the traditional phosphate or borate buffering solution, the biological buffering agent can better maintain the cell morphology and has good stability, so that the analysis of the blood cells of the beasts can be accurately carried out.
In order to verify the beneficial technical effects of the sheath fluid, a series of experiments are carried out on the performance of the sheath fluid from the aspects of technical parameters such as a buffer system, a defoaming agent, a pH value and the like, and in the application, two groups of animal blood samples are selected for analysis, the two groups of animal blood samples are dogs and cats respectively, in order to check the test accuracy of the sheath fluid of the invention on the animal blood samples, an IDEXX five-classification blood cell analyzer which is accepted in the market and is used for accurately analyzing the animal blood samples and the IDEXX animal sheath fluid are used for analyzing the selected animal blood samples, and the selected animal blood samples are mainly analyzed for leucocytes (WBC), Neutrophils (NEU), Lymphocytes (LYM), Monocytes (MONO), Eosinophils (EOS) and Basophils (BASO) in the animal blood samples, actual measurements are given, and the actual measurements for five-class blood cell analysis of the two species veterinary blood samples of the present application are shown in table one.
Table one: five-differential blood cell analysis of canine and feline blood samples
In order to verify that the N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid in the application has a better effect of maintaining cell morphology, a series of five-classification blood cell analysis is performed on different selected canine blood samples by applying sheath fluids added with different buffer solutions, the comparison analysis is mainly performed on the sheath fluid added with the N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid and the sheath fluid added with disodium hydrogen phosphate-sodium dihydrogen phosphate, and detection analysis data are shown in a table two.
Table two: five-differential blood cell analysis of canine blood samples with sheath fluid supplemented with different buffer solutions
From the second table, it can be seen that the various white blood cell count values of the five-class blood cell analysis of different canine blood samples detected using the sheath fluid supplemented with N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid are closer to the actual measurement values (see the first table). Therefore, the N-tri (hydroxymethyl) methyl-3-aminopropanesulfonic acid has better effect on maintaining cell morphology and good stability.
In the application, in order to facilitate the preparation of the sheath solution, the buffer solution in the invention further comprises a second buffer solution, and the effective buffering pH value range of the common buffer solution is given in the third table. As can be seen from Table III, the pH ranges of N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) and Tris (hydroxymethyl) aminomethane (Tris) are both closest to the optimal pH range for the veterinary blood cell analysis, and therefore, in the present invention, Tris is also included in the buffer solution.
Table three: effective buffering pH value range of different buffers
| Buffer name | Range of pH value |
| N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) (2.0mol/L) | 7.7～9.1 |
| Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (0.2mol/L) | 5.8～8.0 |
| Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (1/15molL) | 4.92～8.18 |
| Dipotassium phosphate-sodium hydroxide buffer (0.05mol/L) | 5.8～8.0 |
| Borax-sodium hydroxide buffer (0.05mol/L) | 9.3～10.1 |
| Tris (0.05mol/L) | 6.8～9.6 |
The buffering agent in chemical engineering is often called as acid-base stabilizer, generally salts, such as strong acid-weak base or weak acid-strong base salts, which gradually release acid or base in the salts to maintain stable acid-base value during reaction or preservation, and the specific principle is that when a certain amount of acid or base is added to some solutions, the buffering agent has the effect of hindering the pH change of the solutions, thereby playing a buffering role, but when a single buffering solution is used for adjusting the pH value, since only one substance participates in the reaction, the reaction is rapid, and it is not easy to control when adjusting the pH value range, therefore, in the scheme of the present application, it is preferable to select Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid as the first buffering agent, and also add a small amount of buffering agent as the second buffering agent, and the combination of the first buffering agent and the second buffering agent has a better stabilizing effect on the adjustment of the pH value of the solutions, as can be seen from the experimental data provided in table four, the hydrochloric acid solutions having the same concentration and quality are added to the sheath fluid to be adjusted having different types of buffers, and the pH of the sheath fluid to be adjusted is in the reference range of 9.0 to 11.0, wherein the pH of the sheath fluid containing Tris and N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid is stabilized at 8.3 after the reaction with hydrochloric acid, and therefore, the buffer combined with Tris and N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid has a better buffering effect, and is favorable for adjusting the pH range of the sheath fluid to a desired range. In the present application, it is further preferred that the buffer is a mixed buffer solution of Tris and N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid.
Table four: analysis of buffer Capacity of solutions containing the same hydrochloric acid concentration with different buffers
In this application, the defoaming agent has been added to this sheath liquid, and the main effect can eliminate or reduce the bubble that sheath liquid parcel sample got into sheath flow cell counting in-process well. The polyether defoamer in the defoamer has the characteristics of no toxicity, environmental protection, no stimulation and easy dispersion in water, so the polyether defoamer is preferably used in the defoamer. The polyether antifoaming agent is classified into GP type polyether antifoaming agent, GPE type antifoaming agent and GPEs type antifoaming agent, and three antifoaming agents are described in detail below: 1) GP type polyether defoamer: the defoaming agent is prepared by polymerization of propylene oxide or a mixture of ethylene oxide and propylene oxide by using glycerol as an initiator, has low solubility in water, is not suitable for defoaming in a water system, can be used in a culture medium, and has better foam inhibition effect than defoaming effect; 2) GPE type antifoam agent: ethylene oxide is added at the tail end of a polypropylene glycol chain segment of the GP-type defoaming agent to form polyoxyethylene oxypropylene glycerin with a hydrophilic group at the chain end, however, the addition amount of the ethylene oxide is 10 percent, 20 percent and … … 50 percent are respectively called GPE10, GPE20, … … GPE50 and the like, and the defoaming agent is easy to dissolve in a medium and can be used for quickly defoaming, but can not be used for inhibiting foam for a long time and has shorter foam inhibition duration; 3) GPES type antifoam agent: at the end of a GPE type defoaming agent chain, hydrophobic stearate is used for sealing a head to form a block copolymer with hydrophobic chains at two ends and a hydrophilic chain in the middle, molecules of the structure are easy to horizontally gather at a gas-liquid interface, the surface activity is strong, the defoaming efficiency is high, and the defoaming effect is good. To achieve less air bubbles when the sheath fluid wraps the sample, the antifoam agent of the present application is preferably a GPES type antifoam agent. Furthermore, the mass percentage content of the GPES defoaming agent is 0.001-0.05%.
Table five: five-classification analysis result of white blood cells of blank samples by GPES antifoaming agent sheath fluid with different concentrations
As can be seen from table five, the higher the mass percentage of the GPES antifoaming agent in the sheath fluid, the lower the count value of each kind of leukocyte of the five kinds of leukocyte in the blank sheath fluid, when the mass percentage of the GPES antifoaming agent in the sheath fluid is greater than or equal to 0.001%, the count range of each kind of leukocyte of the five kinds of leukocyte in the sheath fluid can meet the requirement of analyzing the blood cells of the animal, and when the mass percentage of the GPES antifoaming agent in the sheath fluid is greater than or equal to 0.05%, the count value of each kind of leukocyte of the five kinds of leukocyte in the blank sheath fluid is constantly stabilized to 0, but experiments prove that the higher the mass percentage of the GPES antifoaming agent can affect the osmotic pressure of the solution or bring other impurities, and therefore, when the mass percentage of the GPES antifoaming agent in the sheath fluid is controlled between 0.001% and 0.05%, the detection effect is the best.
In this application, still to not adding the sheath liquid of defoaming agent, add non-GPES type defoaming agent sheath liquid and add GPES type defoaming agent sheath liquid and carried out five categorised leucocyte analyses, in the experiment, 1) not adding the sheath liquid of defoaming agent: the components of the anti-foaming agent comprise purified water, ethylene glycol phenyl ether, Tris, N-trihydroxymethyl-3-aminopropanesulfonic acid (TAPS), sodium chloride and hydrochloric acid, and 2) non-GPES type defoaming agent sheath fluid: the components of the defoaming agent comprise purified water, ethylene glycol phenyl ether, Tris, N-trihydroxymethyl-3-aminopropanesulfonic acid (TAPS), sodium chloride, hydrochloric acid, lauryl phenylacetate and 3) GPES type defoaming agent sheath fluid: the components of the kit comprise purified water, ethylene glycol phenyl ether, Tris, N-trihydroxymethyl-3-aminopropanesulfonic acid (TAPS), sodium chloride, hydrochloric acid and triton-X, five-classification leukocyte analysis is respectively carried out on three types of blank sheath fluid, and test data are shown in a table six.
Table six: five-classification analysis result of white blood cells of blank samples by different kinds of antifoaming agent sheath fluids
As can be seen from the test data in table six, when the GPES defoaming agent was added to the sheath fluid, the number of bubbles in the sheath fluid was the smallest, and the number of counts of each type of white blood cells in the five types of white blood cells was the lowest, so that the GPES defoaming agent added to the sheath fluid had good cleanliness, which was advantageous for improving the accuracy of the blood analysis of the animals.
In the application, the bacteriostatic agent is preferably ethylene glycol phenyl ether, and the characteristics of the ethylene glycol phenyl ether are as follows: toxicity-low toxicity, no allergy, biological buffer solution is used as a substitute for highly toxic sodium azide; the phenoxyethanol has no drug resistance, has specific bacteriostatic activity on pseudomonas aeruginosa, is used as another bacteriostatic agent of chlorhexidine acetate, and pseudomonas aeruginosa can tolerate 0.2 percent of chlorhexidine acetate; in addition, ethylene glycol phenyl ether, as a standard compound for preservatives, does not generally release formaldehyde.
In the present application, as a preferred embodiment, the osmotic pressure regulator is an inorganic salt; the inorganic salt is further preferably sodium chloride; the mass concentration of each component in the sheath fluid is as follows: the buffer solution is 0.5 g/L-1.0 g/L, and the ethylene glycol phenyl ether is 3 g/L-8 g/; the mass concentration of the sodium chloride in the sheath fluid is preferably 8g/L to 15 g/L.
In the application, a pH value regulator is added into the sheath fluid to regulate the pH value of the sheath fluid, the pH value regulator is preferably hydrochloric acid, when the scheme of the invention is applied to analyzing the blood cells of the beasts, the optimal pH value range analysis is also carried out, the blood sample of the beasts in the experiment is preferably a dog blood sample, and the actual measurement value of the five-differential leukocyte analysis of the blood sample refers to the table I. The components in the first group of sheath fluid embodiments provided by the present application are: the sheath liquid for the 1L veterinary hemocyte analyzer comprises 5.5g of ethylene glycol phenyl ether, 0.25g of Tris, 0.55g of N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid, 0.002g of GPES defoaming agent, 11g of sodium chloride, and the balance of hydrochloric acid and pure water, wherein the hydrochloric acid is used for adjusting the pH value, and the pH value of the sheath liquid is 7.8-8.8. In the first group of sheath fluid examples, the effect of different pH values on the accuracy of the test of the veterinary blood cells is shown in table seven.
TABLE VII: influence of sheath fluids with different pH values on testing precision of five-classification blood cells of dog blood samples
The components in the second group of sheath fluid embodiments provided by the present application are: the sheath liquid for the 1L veterinary hemocyte analyzer comprises 5.5g of ethylene glycol phenyl ether, 0.25g of Tris, 0.55g of N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid, 0.01g of GPES defoaming agent, 11g of sodium chloride, and the balance of hydrochloric acid and pure water, wherein the hydrochloric acid is used for adjusting the pH value, and the pH value of the sheath liquid is 7.8-8.8. In the second group of sheath fluid examples, the effect of different pH values on the accuracy of the canine blood cell test is shown in Table eight.
Table eight: influence of sheath fluids with different pH values on testing precision of five-classification blood cells of dog blood samples
From the seventh table and the eighth table, when the pH value range of the sheath fluid is 7.8-8.8, the five-classification leukocyte test value of the animal blood sample is closest to the actual five-classification leukocyte measurement value of the animal blood sample, and therefore, the sheath fluid of the present invention has more accurate analysis on the animal blood cells within the pH value range.
The technical scheme provided by the invention has a better detection effect in the detection of the blood cells of the beasts, and the technical scheme is suitable for various beasts such as dogs, cats, pigs, horses, rabbits, sheep, monkeys and the like.
Table nine: and (3) analyzing and comparing the veterinary blood sample with the human blood cell sheath fluid analysis reagent for veterinary and human blood cells:
it can be known from table nine that the sheath fluid for the animal blood cell analyzer of the present invention has a more accurate detection effect when used for animal blood cell analysis, and the detection value is closer to the actual measurement value of the animal blood sample, therefore, the sheath fluid of the present invention has a more accurate measurement value when used for leukocyte detection and analysis of animal blood cells than the conventional sheath fluid used for human blood leukocyte analysis.
The application also provides a preparation method of the sheath fluid for the veterinary blood cell analyzer, which comprises the following steps: s1, sequentially dissolving a buffering agent, a bacteriostatic agent, an antifoaming agent and an osmotic pressure regulator into pure water, and performing constant volume to obtain a mixed solution; s2, adjusting the pH value of the mixed solution in the step S1 by using a pH value regulator, and controlling the pH value range of the mixed solution to be 7.8-8.8 to obtain a sheath liquid for the animal hemocyte analyzer; s3, filtering the sheath liquid in the step S2 by a 0.22 μm polypropylene membrane, and storing the sheath liquid in a sealed manner by using a container, wherein the container comprises a packaging cover, preferably, a deoxidizer is arranged on the packaging cover, the content of dissolved oxygen in the reagent is increased after the packaging cover is actually unsealed and during transportation, and the deoxidizer on the packaging cover can consume the dissolved oxygen in the reagent to a certain extent and reduce the generation of bubbles caused by the existence of the dissolved oxygen in the test process. Wherein the deoxidizer is an organic matrix deoxidizer, and the concentration of the organic matrix deoxidizer is preferably 0.01 g/L.
The sheath fluid for the veterinary hemocyte analyzer has the beneficial effects that the sheath fluid comprises the following components: the antibacterial agent comprises a buffer solution, a bacteriostatic agent, a defoaming agent, an osmotic pressure regulator, a pH value regulator and pure water, wherein the buffer solution comprises N-tri (hydroxymethyl) methyl-3-aminopropanesulfonic acid; the pH value range of the sheath liquid is controlled to be 7.8-8.8. Compared with the traditional phosphate buffer solution or borate buffer solution, the N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid can better maintain the cell morphology, the invention also provides a preparation method and a storage mode of the sheath liquid, a deoxidizer is placed on the packaging cover during storage, the addition of the defoamer and the use of the deoxidizer both reduce the foaming degree of the sheath liquid and improve the test precision of the blood cells of the animals.
The present application has been described in connection with only the presently preferred embodiments with the understanding that the present disclosure is not to be considered as limiting, and the present application is not limited to the examples described above, but rather, it is to be understood that changes, modifications, additions or substitutions that are within the spirit and scope of the application by one of ordinary skill in the art are included.
Claims (10)
1. A sheath liquid for a veterinary hemocyte analyzer is characterized by comprising the following components: the antibacterial agent comprises a buffer solution, a bacteriostatic agent, a defoaming agent, an osmotic pressure regulator, a pH value regulator and pure water, wherein the buffer solution comprises N-tri (hydroxymethyl) methyl-3-aminopropanesulfonic acid; the pH value range of the sheath liquid is controlled to be 7.8-8.8.
2. The sheath fluid for a veterinary blood cell analyzer according to claim 1, wherein the buffer solution further comprises Tris.
3. The sheath fluid for a veterinary use blood cell analyzer according to claim 2, wherein the antifoaming agent is a GPES type antifoaming agent, the bacteriostatic agent is ethylene glycol phenyl ether, and the osmotic pressure regulator is an inorganic salt.
4. The sheath fluid for a veterinary use blood cell analyzer according to claim 3, wherein the mass concentration of each of the components in the sheath fluid is as follows: the buffer solution is 0.5-1.0 g/L, the ethylene glycol phenyl ether is 3-8 g/L, and the GPES defoaming agent is 0.001-0.05 g/L.
5. The sheath fluid for a veterinary blood cell analyzer according to claim 4, wherein the inorganic salt is sodium chloride.
6. The sheath fluid for a veterinary blood cell analyzer according to claim 5, wherein in 1L of the sheath fluid,
the balance of pH value regulator and pure water;
the pH value of the sheath liquid is 7.8-8.8.
7. A method for preparing a sheath fluid for a blood cell analyzer for animals, which is applied to any one of claims 1 to 6, comprising the steps of:
s1, sequentially dissolving the buffer solution, the bacteriostatic agent, the defoaming agent and the osmotic pressure regulator into pure water, and performing constant volume to obtain a mixed solution;
s2, adjusting the pH value of the mixed solution in the step S1 by using a pH value regulator, and controlling the pH value range of the mixed solution to be 7.8-8.8 to obtain a sheath liquid for the animal hemocyte analyzer;
s3, filtering the sheath liquid in the step S2 by adopting a 0.22 mu m polypropylene membrane, and storing the sheath liquid in a sealed way by using a container.
8. The method according to claim 7, wherein the container includes a sealing cap, and a deoxidizer is placed on the sealing cap.
9. The method for preparing a sheath fluid for a veterinary blood cell analyzer according to claim 8, wherein the deoxidizer is an organic matrix deoxidizer.
10. The method for preparing a sheath fluid for a veterinary blood cell analyzer according to claim 9, wherein the organic substrate deoxidizer is present in a concentration of 0.01 g/L.
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| US20180172562A1 (en) * | 2015-03-31 | 2018-06-21 | Eiken Kagaku Kabushiki Kaisha | Preservative solution for heme protein, and method for stabilizing heme protein |
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| US20030219752A1 (en) * | 1995-12-07 | 2003-11-27 | Diversa Corporation | Novel antigen binding molecules for therapeutic, diagnostic, prophylactic, enzymatic, industrial, and agricultural applications, and methods for generating and screening thereof |
| US20070177146A1 (en) * | 2006-01-31 | 2007-08-02 | Sysmex Corporation | Sheath liquid for particle analyzer |
| US20180172562A1 (en) * | 2015-03-31 | 2018-06-21 | Eiken Kagaku Kabushiki Kaisha | Preservative solution for heme protein, and method for stabilizing heme protein |
| US20170318801A1 (en) * | 2015-06-30 | 2017-11-09 | Sysmex Corporation | Method of preserving cells |
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Application publication date: 20210129 |