CN111781047A - Hemolysin for blood analysis and preparation method and reagent thereof - Google Patents
Hemolysin for blood analysis and preparation method and reagent thereof Download PDFInfo
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- 108010006464 Hemolysin Proteins Proteins 0.000 title claims abstract description 70
- 239000003228 hemolysin Substances 0.000 title claims abstract description 70
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 72
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 48
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 48
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 48
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- 239000001103 potassium chloride Substances 0.000 claims abstract description 24
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- 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
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- 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
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Abstract
The invention discloses hemolysin for blood analysis and a preparation method and a reagent thereof, wherein the hemolysin comprises octadecyl sodium sulfate, ethanol, citric acid, sodium chloride, potassium chloride and sodium bicarbonate.
Description
Technical Field
The invention relates to the technical field of blood analysis, in particular to hemolysin for blood analysis, a preparation method thereof and a reagent.
Background
Blood cell analysis is of great value for the development of scientific research, prevention, diagnosis and treatment of disease, ranging from the initial study of cell morphology, size, number to the recent antigenic components on cell surfaces. The traditional cell immunity and non-specific immunity detection technology can not carry out multi-parameter and high-sensitivity analysis on the size, the shape, the plasma membrane and the internal structure of a target cell from a single cell level, a flow cytometer can quickly measure, sort, store and display a series of important characteristic parameters in biophysical and biochemical aspects of dispersed cells suspended in liquid, and can sort out specified cell subsets according to preselected parameter ranges, and the traditional cell immunity and non-specific immunity detection technology is currently applied to the fields of clinical medicine and basic medicine research such as immunology, hematology, oncology, cytogenetics, cytobiology, biochemistry and the like, such as lymphocyte and subgroup analysis of cell surface molecules, intracellular cytokine analysis, leukemia immunity typing, tumor drug-resistant related protein analysis, autoimmune disease related HLA antigen analysis, cross-matching type in transplantation immunity, Mitochondrial membrane potential, apoptosis, intracellular calcium ion concentration, neutrophil function analysis, and the like.
Because the majority of the hemocytes which play immune functions in vivo are leukocytes, the erythrocytes which have the highest content in human blood are erythrocytes, and the interference effect on the analysis of target cells needs to be eliminated, the structure and function of the leukocytes are prevented from being damaged when the hemolysis of the erythrocytes is carried out, and membrane proteins on the surfaces of the leukocytes are reserved, so the requirement on the hemolysis is very high, most of hemolysins for the blood analysis of the existing flow cells are hypotonic hemolysins for the blood analysis, namely solutions lower than osmotic pressure, and the hemolysins for the blood analysis of isotonic solution is solutions equal to the osmotic pressure, and the erythrocytes are considered to be in isotonic solutions and can keep normal forms (not shrink and not be hemolyzed), but some molecules can freely pass through cell membranes to hemolyze the erythrocytes, and the cell membranes are ruptured by utilizing the difference of the osmotic pressure inside and outside the cells, the hemolysis reaction is achieved by changing the physical structure of the red blood cells. Some chemicals such as drugs, bacterial toxins, snake venom, etc. bind to ferrous hemoglobin in erythrocytes and become methemoglobin, which changes the structure of erythrocytes and leads to hemolytic reactions.
At present, most hemolysins used for blood analysis in China are imported original reagents, the cost is high, the purchase channel is complicated and the like, normal use of domestic hospitals cannot be met in time, a formula of the hemolysin used for blood analysis and an instrument have a certain relation, different hemolysins used for blood analysis are used by different instruments, the same hemolysin used for blood analysis has different display effects on different instruments, the main reason is that leucocyte forms after the hemolysin used for blood analysis is cracked are different, and the difference of lymph, mononuclear and granulocyte grouping can be caused by combining the optical path design of FSC/SSC. Many hemolysins for blood analysis in the market at present are only suitable for a certain type of flow cytometry, and cannot achieve the effect of a hemolysin general multi-type flow cytometer.
Therefore, the invention discloses hemolysin for blood analysis, a preparation method and a reagent thereof, which have the advantages of simple preparation, high detection precision and wide application range.
Disclosure of Invention
In view of the above problems, the present invention provides hemolysin for blood analysis, and a preparation method and a reagent thereof, and the technical scheme adopted by the present invention is as follows:
the hemolysin for blood analysis comprises, per liter, 0.5-1.5 g of sodium octadecyl sulfate, 0.02-0.09 g of ethanol, 2-3 g of citric acid, 1-10 g of sodium chloride, 5.1-5.5 g of potassium chloride and 0.1 × 10g of sodium bicarbonate-3～0.9×10-3g。
Furthermore, each liter of the hemolysin for blood analysis comprises 0.8-1.5 g of sodium octadecyl sulfate, 0.04-0.09 g of ethanol, 2.5-3 g of citric acid, 3-10 g of sodium chloride, 5.2-5.5 g of potassium chloride and 0.3 × 10.10 g of sodium bicarbonate-3～0.9×10-3g。
Furthermore, each liter of the hemolysin for blood analysis comprises 1.0-1.5 g of sodium octadecyl sulfate, 0.06-0.09 g of ethanol, 2.8-3 g of citric acid, 7-10 g of sodium chloride, 5.4-5.5 g of potassium chloride and 0.6 × 10.10 g of sodium bicarbonate-3～0.9×10-3g。
Furthermore, each liter of the hemolysin for blood analysis comprises 1.4-1.5 g of sodium octadecyl sulfate, 0.07-0.09 g of ethanol, 2.9-3 g of citric acid, 9-10 g of sodium chloride, 5.4-5.5 g of potassium chloride and 0.8 × 10.10 g of sodium bicarbonate-3～0.9×10-3g。
Further, a method for preparing hemolysin for blood analysis, comprising the steps of:
s01, weighing octadecyl sodium sulfate, ethanol, citric acid, sodium chloride, potassium chloride and sodium bicarbonate according to the content of each liter of the hemolysin for blood analysis;
step S02, adding the sodium octadecyl sulfate, citric acid, sodium chloride and potassium chloride components weighed in the step S01 into M1g, fully dissolving in water to obtain a primary mixed solution;
step S03, adding ethanol into the mixed solution prepared in step S02, and adding M2g, water is added to a constant volume of 1 liter, and a premix is prepared;
step S04, adding the sodium bicarbonate weighed in the step S01 into the premix prepared in the step S03, and adjusting the pH value of the premix to 7.4-7.5;
and S05, filtering the mixed solution prepared in the step S04 by using a microporous filter membrane with the aperture of 0.1-0.2 mu m, and preparing the hemolysin for blood analysis.
Further, the hemolysin for blood analysis prepared according to the hemolysin preparation method for blood analysis is included.
Further, an asymmetric cyanine dye, the hemolysin for blood analysis as described in any one of claims 1 to 4 and claim 6, is included.
Furthermore, each liter of hemolysin for blood analysis comprises 0.2-0.5 g of asymmetric cyanine dye.
Compared with the prior art, the invention has the following beneficial effects:
(1) the hemolysin for blood analysis prepared by the invention can change the cell structure without destroying the shape, structure and function of the leucocyte, and has the advantages of high detection precision, less interference impurities, low use cost and wide application range.
(2) The hemolysin for blood analysis prepared by the invention has the beneficial effect of reaching the general multi-class flow cytometry.
(3) The hemolysin for blood analysis obtained by the preparation method can realize accurate detection of the nucleated red blood cells, the nucleic acids and other types of white blood cells in blood in one channel.
(4) The reagent for blood detection is prepared by skillfully adding the asymmetric cyanine dye into the hemolysin for blood analysis, and in the process of analyzing blood, the prepared hemolysin for blood analysis not only modifies the nucleic acid which fixes the nucleated red blood cells and the basophils for dyeing, but also can detect the two types of cells through multi-angle light scattering, and also ensures the counting accuracy of the basophils.
(5) The hemolysin for blood analysis prepared by the invention is a key reagent for detecting leukocyte antigens and analyzing cellular immune functions, and the leukocyte populations are divided into lymphocytes, monocytes, neutrophils and fragments by means of forward angle and lateral angle astigmatism on a flow cytometer according to the cell size and granularity, after a sample is processed, each leukocyte population is obviously partitioned, the cell fragments are few, the leukocyte membrane protein is not damaged, the original biological activity can be kept, and erythrocytes can be completely lysed.
The hemolysin for blood analysis, the preparation method thereof and the reagent have high practical value, economic value and popularization value in the technical field of blood analysis.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope of protection, and it is obvious for those skilled in the art that other related drawings can be obtained according to these drawings without inventive efforts.
FIG. 1 is a graph showing the comparison of the results of the white blood cell count test using the reagent of example 4;
FIG. 2 is a graph showing the experimental results of absorbance of hemoglobin derivative measured by the reagent test described in example 4 of the present invention;
FIG. 3 is a graph showing the comparison of the results of the erythrocyte count test using the reagent of example 4 of the present invention.
Detailed Description
To further clarify the objects, technical solutions and advantages of the present application, the present invention will be further described with reference to the accompanying drawings and examples, and embodiments of the present invention include, but are not limited to, the following examples. 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.
Example 1
Specifically, a reagent for blood analysis includes hemolysin and a staining solution for blood analysis.
In this embodiment, the method for preparing hemolysin for blood analysis includes the following steps:
step S01, weighing 0.5g sodium octadecyl sulfate, 0.02g ethanol, 2g citric acid, 1g sodium chloride, 5.1g potassium chloride and 0.1 × 10g sodium bicarbonate-3g, standby;
step S02, adding 0.5g of sodium octadecyl sulfate, 2g of citric acid, 1g of sodium chloride and 5.1g of potassium chloride which are weighed in the step S01 into 600g of water for fully dissolving to obtain a primary mixed solution;
step S03, adding 0.02g of ethanol into the primary mixed liquid prepared in the step S02, and adding 390g of water to a constant volume of 1 liter to prepare a premixed liquid;
step S04, adding sodium bicarbonate 0.1 × 10-3g, stabilizing the pH value of the premix in the premix prepared in the step S03 at 7.4 to obtain a mixed solution;
and S05, filtering the mixed solution prepared in the step S04 by using a microfiltration membrane with the pore diameter of 0.2 mu m, and preparing the hemolysin for blood analysis.
In this embodiment, the reagent for blood analysis includes the following steps:
step S01, weighing 0.3g of asymmetric cyanine dye for later use;
and step S02, adding the asymmetric cyanine dye weighed in the step S01 into the prepared hemolysin for blood analysis.
Example 2
A reagent for blood analysis, comprising hemolysin and a staining solution for blood analysis.
In this example, each liter of hemolysin for blood analysis comprises the following components of 0.8g of sodium octadecyl sulfate, 0.04g of ethanol, 2.5g of citric acid, 3g of sodium chloride, 5.2g of potassium chloride and 0.3 × 10g of sodium bicarbonate-3g。
In this embodiment, the method for preparing hemolysin for blood analysis includes the following steps:
step S01, weighing 0.8g sodium octadecyl sulfate, 0.04g ethanol, 2.5g citric acid, 3g sodium chloride, 5.2g potassium chloride and 0.3 × 10g sodium bicarbonate-3g, standby;
step S02, adding 0.8g of sodium octadecyl sulfate, 2.5g of citric acid, 3g of sodium chloride and 5.2g of potassium chloride which are weighed in the step S01 into 600g of water for fully dissolving to obtain a primary mixed solution;
step S03, adding 0.04g of ethanol into the primary mixed liquid prepared in the step S02, and adding 390g of water to a constant volume of 1 liter to prepare a premixed liquid;
step S04, adding sodium bicarbonate 0.3 × 10-3g, stabilizing the pH value of the premix in the premix prepared in the step S03 at 7.4 to obtain a mixed solution;
and S05, filtering the mixed solution prepared in the step S04 by using a microfiltration membrane with the pore diameter of 0.2 mu m, and preparing the hemolysin for blood analysis.
In this embodiment, the reagent for blood analysis includes the following steps:
step S01, weighing 0.3g of asymmetric cyanine dye for later use;
and step S02, adding the asymmetric cyanine dye weighed in the step S01 into the prepared hemolysin for blood analysis.
Example 3
A reagent for blood analysis, comprising hemolysin and a staining solution for blood analysis.
In this example, each liter of the hemolysin for blood analysis comprises the following components of 1.0g of sodium octadecyl sulfate, 0.06g of ethanol, 2.8g of citric acid, 7g of sodium chloride, 5.4g of potassium chloride and 0.6 × 10g of sodium bicarbonate-3g。
In this embodiment, the method for preparing hemolysin for blood analysis includes the following steps:
step S01, weighing 1.0g of sodium octadecyl sulfate, 0.06g of ethanol, 2.8g of citric acid, 7g of sodium chloride, 5.4g of potassium chloride and 0.6 × 10g of sodium bicarbonate-3g, standby;
step S02, adding 1.0g of sodium octadecyl sulfate, 2.8g of citric acid, 7g of sodium chloride and 5.4g of potassium chloride which are weighed in the step S01 into 600g of water for fully dissolving to obtain a primary mixed solution;
step S03, adding 0.06g of ethanol into the primary mixed liquid prepared in the step S02, and then adding 390g of water to a constant volume of 1 liter to prepare a premixed liquid;
step S04, adding sodium bicarbonate 0.6 × 10-3g, stabilizing the pH value of the premix in the premix prepared in the step S03 at 7.4 to obtain a mixed solution;
and S05, filtering the mixed solution prepared in the step S04 by using a microfiltration membrane with the pore diameter of 0.1 mu m, and preparing the hemolysin for blood analysis.
In this embodiment, the reagent for blood analysis includes the following steps:
step S01, weighing 0.4g of asymmetric cyanine dye for later use;
and step S02, adding the asymmetric cyanine dye weighed in the step S01 into the prepared hemolysin for blood analysis.
Example 4
A reagent for blood analysis, comprising hemolysin and a staining solution for blood analysis.
In this example, each liter of the hemolysin for blood analysis comprises the following components of 1.4g of sodium octadecyl sulfate, 0.07g of ethanol, 2.9g of citric acid, 9g of sodium chloride, 5.4g of potassium chloride and 0.8 × 1g of sodium bicarbonate0-3g。
In this embodiment, the method for preparing hemolysin for blood analysis includes the following steps:
step S01, weighing 1.4g sodium octadecyl sulfate, 0.07g ethanol, 2.9g citric acid, 9g sodium chloride, 5.4g potassium chloride and 0.8 × 10g sodium bicarbonate-3g, standby;
step S02, adding 1.4g of sodium octadecyl sulfate, 2.9g of citric acid, 9g of sodium chloride and 5.4g of potassium chloride which are weighed in the step S01 into 600g of water for fully dissolving to obtain a primary mixed solution;
step S03, adding 0.07g of ethanol into the primary mixed liquid prepared in the step S02, and adding 390g of water to a constant volume of 1 liter to prepare a premixed liquid;
step S04, adding sodium bicarbonate 0.8 × 10-3g, stabilizing the pH value of the premix in the premix prepared in the step S03 at 7.4 to obtain a mixed solution;
and S05, filtering the mixed solution prepared in the step S04 by using a microfiltration membrane with the pore diameter of 0.2 mu m, and preparing the hemolysin for blood analysis.
In this embodiment, the reagent for blood analysis includes the following steps:
step S01, weighing 0.4g of asymmetric cyanine dye for later use;
and step S02, adding the asymmetric cyanine dye weighed in the step S01 into the prepared hemolysin for blood analysis.
Comparative example
This comparative example provides an inlet genuine reagent for blood analysis.
Accuracy determination experiment: under the same experimental environment, 15 blood samples were taken and tested and analyzed by comparing the reagent provided in example 4 with the existing commercial imported reagent, and the analysis results are shown in the following table.
Table 1:
table 2:
it can be seen from tables 1 and 2 that the reagent provided by the embodiment of the present invention, which only contains a hemolysin and a staining solution for blood analysis, can simultaneously and accurately detect the contents of nucleated red blood cells and basophils in blood, has a detection and analysis effect very high similar to that of the existing commercially available imported original reagent, can be used instead of the original reagent, so as to reduce the development cost of blood analysis and the reagent use cost of each medical institution, and solve the dependency of each medical institution on the imported reagent.
The above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but all changes that can be made by applying the principles of the present invention and performing non-inventive works on the basis of the principles shall fall within the scope of the present invention.
Claims (8)
1. The hemolysin for blood analysis is characterized by comprising, per liter, 0.5-1.5 g of sodium octadecyl sulfate, 0.02-0.09 g of ethanol, 2-3 g of citric acid, 1-10 g of sodium chloride, 5.1-5.5 g of potassium chloride and 0.1 × 10.10 g of sodium bicarbonate-3～0.9×10-3g。
2. The hemolysin for blood analysis according to claim 1, wherein each liter of the hemolysin for blood analysis comprises 0.8-1.5 g of sodium octadecyl sulfate, 0.04-0.09 g of ethanol, 2.5-3 g of citric acid, 3-10 g of sodium chloride, 5.2-5.5 g of potassium chloride, and 0.3 × 10g of sodium bicarbonate-3～0.9×10-3g。
3. A hemolysin according to claim 2, characterized in that said hemolysin for blood analysis comprises per liter the following components1.0 to 1.5g of sodium octadecyl sulfate, 0.06 to 0.09g of ethanol, 2.8 to 3g of citric acid, 7 to 10g of sodium chloride, 5.4 to 5.5g of potassium chloride and 0.6 × 10g of sodium bicarbonate-3～0.9×10-3g。
4. The hemolysin for blood analysis according to claim 3, wherein each liter of the hemolysin for blood analysis comprises 1.4-1.5 g sodium octadecyl sulfate, 0.07-0.09 g ethanol, 2.9-3 g citric acid, 9-10 g sodium chloride, 5.4-5.5 g potassium chloride, and 0.8 × 10g sodium bicarbonate-3～0.9×10-3g。
5. A method for preparing hemolysin for blood analysis, which is characterized by comprising the following steps:
s01, weighing octadecyl sodium sulfate, ethanol, citric acid, sodium chloride, potassium chloride and sodium bicarbonate according to the content of each liter of the hemolysin for blood analysis;
step S02, adding the sodium octadecyl sulfate, citric acid, sodium chloride and potassium chloride components weighed in the step S01 into M1g, fully dissolving in water to obtain a primary mixed solution;
step S03, adding ethanol into the mixed solution prepared in step S02, and adding M2g, water is added to a constant volume of 1 liter, and a premix is prepared;
step S04, adding the sodium bicarbonate weighed in the step S01 into the premix prepared in the step S03, and adjusting the pH value of the premix to 7.4-7.5;
and S05, filtering the mixed solution prepared in the step S04 by using a microporous filter membrane with the aperture of 0.1-0.2 mu m, and preparing the hemolysin for blood analysis.
6. A hemolysin for blood analysis comprising the hemolysin for blood analysis obtained by the hemolysin preparation method for blood analysis according to claim 5.
7. A reagent for blood analysis comprising an asymmetric cyanine dye, the hemolysin for blood analysis as described in any one of claims 1 to 4 and claim 6.
8. The reagent for blood analysis according to claim 7, wherein the hemolysin for blood analysis comprises 0.2 to 0.5g of asymmetric cyanine dye per liter.
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