CN104297134A - Hemolytic agent and application thereof as well as classifying and counting method for white blood cells - Google Patents
Hemolytic agent and application thereof as well as classifying and counting method for white blood cells Download PDFInfo
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- CN104297134A CN104297134A CN201410620796.0A CN201410620796A CN104297134A CN 104297134 A CN104297134 A CN 104297134A CN 201410620796 A CN201410620796 A CN 201410620796A CN 104297134 A CN104297134 A CN 104297134A
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Abstract
The invention relates to the technical field of medical examination and discloses a hemolytic agent and application thereof as well as a classifying and counting method for white blood cells. The hemolytic agent contains one or more than two kinds of cationic surfactants, one or more than two kinds of small molecule alcohols, one or more than two kinds of buffer solutions, one or more than two kinds of soluble inorganic salts and the balance of water. According to the invention, components of the hemolytic agent are simplified; the novel hemolytic agent suitable for classifying and counting basophilic granulocyte and other white blood cells is prepared by adopting the raw materials which are proper and easy to obtain; compared with the current hemolytic agent product with more complex components, the hemolytic agent disclosed by the invention is more convenient and quicker for use; the hemolysis time only needs 5-20s, the classifying and the counting for the basophilic granulocyte and the other white blood cells can be realized by combining the hemolytic agent with an impedance analysis measuring instrument without special requirement on the temperature, and the accuracy is higher.
Description
Technical field
The present invention relates to technical field of medical examination, be specifically related to a kind of hemolytic agent and apply and Arneth's count method.
Background technology
Leucocyte is also known as white blood cell, and one of blood cell component is colourless round cell, more bigger than red blood cell, has nucleus.Neutrophil cell, eosinophil, basophilic granulocyte, lymphocyte and monocyte five kinds can be divided into.Body be inflamed or disease time, leukocyte count often changes, therefore leukocyte analysis is one of test item the most often adopted in medical laboratory's blood test.White blood count and differential is not only all significant to the diagnosis of disease, antidiastole, observation of curative effect and prognostic analysis, and also has certain value to the assessment of health status.
Basophilic granulocyte size in leucocyte is about 10 ~ 15um, rounded or oval, nucleus is about 1:2 ~ 1:3 with the ratio of tenuigenin, karyolobism shape, is often covered by particle, and nuclear chromatin is assembled, without kernel, tenuigenin is containing thick, fine and close, darkviolet or black particle, and in healthy human body blood, basophilic granulocyte accounts for greatly 0.5 ~ 1% of total white blood cells, and basophilia sees chronic myelocytic leukemia, Hodgkin's disease, metastasis of cancer, plumbous bismuthosis etc.
Adopting Rui Shi reagent to dye to sample and carry out cervical arthroplasty is leucocyte five differential count " goldstandard ", but improving constantly of clinical detection being required along with people, and this consuming time, loaded down with trivial details standard method is more and more not suitable with clinical detection of today.Along with the development of medical blood, various types of cellanalyzer coordinates the detection method of special agent (as hemolytic agent etc.) to be widely used at home, these full automatic instruments are first by special agent process red blood cell (RBC), then adopt the counting principle of streaming technology or electrical impedance, leucocyte is divided into variety classes by the fluorescence information of being launched by the different organelle of acquisition leucocyte and scattered light information.
But because inhomogeneous leukocytic volume possibility difference is little, this method of theory of electrical impedance is just by leucocyte simple classification lymphoblast, and monocyte and granulocyte, can not distinguish basophilic granulocyte and counting.Also the instrument had uses light scattering principle, such as patent US3740143, making optically can by lymphocyte, monocyte, neutrophil leucocyte, eosinophil is distinguished and counting, but due to basophilic granulocyte negligible amounts, this method well can not detect basophilic granulocyte.
The multiple reagent for leukocyte differential count is disclosed, as CN2011100744709 relates to a kind of hemolytic agent for leucocyte five differential count and purposes in prior art.Described hemolytic agent is made up of hemolytic agent A and hemolytic agent B.Hemolytic agent A is made up of polyoxyethylene-type nonionic surfactant, organic acid, quaternary cationics cosolvent, stabilizing agent and osmotic pressure regulator; Hemolytic agent B is made up of alkaline, inorganic salts and osmotic pressure regulator.Leucocyte can be divided into lymphocyte, monocyte, eosinophil, neutrophil cell and basophilic granulocyte by this hemolytic agent, obtains all kinds of quantity of leucocyte.
Although the technical scheme of above-mentioned patent notes can effectively distinguish all kinds of leucocyte, but distinguish basophilic granulocyte and other leucocytes for only needing, the hemolytic agent formula that it adopts is comparatively complicated loaded down with trivial details, component is more, and also need on using to come according to certain operation steps, simplicity is poor.
US5196346 (Didier Lefevre) discloses a kind of hemolytic agent measuring leucocyte and basophilic granulocyte, this hemolytic agent uses non-ionics Brij35 and anionic surface active agent, but this hemolytic agent requires high to serviceability temperature, haemolysis speed is slow, and raw material sources are comparatively loaded down with trivial details, still easy not in use.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of hemolytic agent for Arneth's count, such that the haemolysis speed of this hemolytic agent is fast, component is less, easy to use, correctly can distinguish basophilic granulocyte and other leucocytes.
To achieve these goals, the invention provides following technical scheme:
A kind of hemolytic agent, comprises:
One or more cationic surfactants, one or more small molecular alcohols, one or more damping fluids, one or more soluble inorganic salts, surplus is water.
The present invention is directed to the various hemolytic agents that formula is comparatively complicated at present, suitable combination of components is selected to use, develop a kind of novel can be used for and accurately distinguish basophilic granulocyte and other leukocytic hemolytic agents, easy to use and haemolysis speed, compensate for the defect of existing hemolytic agent.
Wherein, as preferably, described hemolytic agent pH value is 1.5-4.5, comprises one or more cationic surfactants that concentration is 0.1g/L-100g/L, one or more soluble inorganic salts that one or more damping fluids that one or more small molecular alcohols that concentration is 0.01g/L-50g/L, concentration are 0.001mol/L-1mol/L, concentration are not more than 30g/L.Surplus is water.
More preferably, described hemolytic agent pH value is 2-3, comprises one or more cationic surfactants that concentration is 1g/L-20g/L, one or more soluble inorganic salts that one or more damping fluids that one or more small molecular alcohols that concentration is 1g/L-20g/L, concentration are 5mmol/L – 200mmol/L, concentration are not more than 20g/L.
As preferably, cationic surfactant described in hemolytic agent of the present invention is selected from quaternary surfactant or/and quaternized pyridinium salt surfactant; Further, most preferably, described quaternary surfactant is selected from DTAC, DTAB, tetradecyl trimethyl ammonium chloride, Tetradecyl Trimethyl Ammonium Bromide, hexadecyltrimethylammonium chloride, cetyl trimethyl ammonium bromide, described quaternized pyridinium salt surfactant is selected from dodecylpyridine chloride ammonium, dococylpyridinium ammonium bromide.
As preferably, described small molecular alcohol is selected from methyl alcohol, ethanol, isopropyl alcohol, benzene oxygen methyl alcohol.
As preferably, described damping fluid is selected from malic acid, succinic acid, phthalic acid, citric acid, tartrate, oxalic acid, formic acid, maleic acid, Gly and their respective salt, and hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid.Buffer concentration of the present invention can also be determined according to the pH value of described hemolytic agent.
As preferably, described soluble inorganic salt is selected from sodium sulphate, potassium sulfate, sodium chloride, potassium chloride.
The present invention also provides a kind of basophilic granulocyte and other leukocytic differential count methods, comprising:
Blood to be measured and hemolytic agent of the present invention are mixed in the counting chamber of theory of electrical impedance blood analyser with volume ratio 1:500-1:100, temperature of reaction 30 ~ 40 DEG C, hemolysis time is 5-20 second, then by the blood sample to be measured handled well successively by counting of gemstones hole, by detecting the change of voltage, obtain basophilic granulocyte and other quantity of leucocyte.
After adopting this hemolytic agent, red blood cell dissolves, and basophilic granulocyte does not shrink, and " bare nucleus " appears in other leucocytes, and Fig. 1 is shown in by schematic diagram.
Hemolytic agent of the present invention is adopted to carry out Arneth's count to blood sample to be measured, after being detected by theory of electrical impedance blood analyser, the data that basophilic granulocyte sum and other total white blood cellses and optical method of obtaining measure do not have significant difference, and result precision is higher.
Thus, present invention also offers the application of hemolytic agent of the present invention in white blood count and differential and preparing the application in white blood count and differential product.As preferably, described white blood count and differential is to basophilic granulocyte and other leukocytic differential counts.
From above technical scheme, the present invention simplifies the component of hemolytic agent, raw material that is suitable, that easily obtain mutually is adopted to prepare the novel hemolytic agent be applicable to basophilic granulocyte and other leukocytic differential counts, comparing the hemolytic agent product that component is comparatively complicated instantly uses more convenient, hemolysis time only needs 5-20s, require to coordinate electrical impedance method surveying instrument to basophilic granulocyte and other leukocytic differential counts without the need to distinct temperature, accuracy is higher.
Accompanying drawing explanation
Figure 1 shows that the form schematic diagram adopting red blood cell, other leucocytes and basophilic granulocyte after hemolytic agent of the present invention.
Embodiment
The invention discloses a kind of hemolytic agent and apply and Arneth's count method, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Preparation method of the present invention and product are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Just a kind of hemolytic agent provided by the present invention and application thereof and Arneth's count method are described further below.
Embodiment 1: hemolytic agent of the present invention
Embodiment 2: hemolytic agent of the present invention
Embodiment 3: hemolytic agent of the present invention
Embodiment 4: hemolytic agent of the present invention
Embodiment 5: hemolytic agent of the present invention
Embodiment 6: accuracy contrast test
Adopt the hemolytic agent of embodiment 1-5 respectively, according to basophilic granulocyte of the present invention and other leukocytic differential count methods, adopt SC-8000A blood analyser (theory of electrical impedance blood analyser) to detect 20 routine blood samples to be measured, method is as follows:
Blood to be measured and hemolytic agent of the present invention are mixed in the counting chamber of theory of electrical impedance blood analyser with volume ratio 1:500-1:100, temperature of reaction 30 ~ 40 DEG C, hemolysis time is 5-20 second, then by the blood sample to be measured handled well successively by counting of gemstones hole, by detecting the change of voltage, obtain basophilic granulocyte and other quantity of leucocyte.
Meanwhile, adopt optical method and microscopy to detect with reference to method above-mentioned healthy blood sample to be measured, the testing result of contrast three, the results are shown in Table 1.
Table 1 testing result
As seen from the above table, hemolytic agent of the present invention is adopted to be detected by electrical impedance, there is no significant difference between the basophilic granulocyte sum obtained and each embodiment of total white blood cells, simultaneously and optical method, Microscopical Method For Detection detect acquisition data and also there is no marked difference, there is higher accuracy.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a hemolytic agent, is characterized in that, comprises:
One or more cationic surfactants, one or more small molecular alcohols, one or more damping fluids, one or more soluble inorganic salts, surplus is water.
2. hemolytic agent according to claim 1, it is characterized in that, pH value is 1.5-4.5, comprise one or more cationic surfactants that concentration is 0.1g/L-100g/L, one or more soluble inorganic salts that one or more damping fluids that one or more small molecular alcohols that concentration is 0.01g/L-50g/L, concentration are 0.001mol/L-1mol/L, concentration are not more than 30g/L, surplus is water.
3. hemolytic agent according to claim 1 or 2, is characterized in that, described cationic surfactant is selected from quaternary surfactant or/and quaternized pyridinium salt surfactant.
4. hemolytic agent according to claim 3, it is characterized in that, described quaternary surfactant is selected from DTAC, DTAB, tetradecyl trimethyl ammonium chloride, Tetradecyl Trimethyl Ammonium Bromide, hexadecyltrimethylammonium chloride, cetyl trimethyl ammonium bromide.
5. hemolytic agent according to claim 3, it is characterized in that, described quaternized pyridinium salt surfactant is selected from dodecylpyridine chloride ammonium, dococylpyridinium ammonium bromide.
6. hemolytic agent according to claim 1 or 2, is characterized in that, described small molecular alcohol is selected from methyl alcohol, ethanol, isopropyl alcohol, benzene oxygen methyl alcohol.
7. hemolytic agent according to claim 1 or 2, it is characterized in that, described damping fluid is selected from malic acid, succinic acid, phthalic acid, citric acid, tartrate, oxalic acid, formic acid, maleic acid, Gly and their respective salt, and hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid.
8. hemolytic agent according to claim 1 or 2, is characterized in that, described soluble inorganic salt is selected from sodium sulphate, potassium sulfate, sodium chloride, potassium chloride.
9. the application of hemolytic agent described in claim 1 in Arneth's count or the application in preparation Arneth's count product.
10. basophilic granulocyte and other leukocytic differential count methods, is characterized in that, comprising:
Described in blood to be measured and claim 1, hemolytic agent is mixed in volume ratio 1:500-1:100 in the counting chamber of theory of electrical impedance blood analyser, temperature of reaction 30 ~ 40 DEG C, hemolysis time is 5-20 second, then by the blood sample to be measured handled well successively by counting of gemstones hole, by detecting the change of voltage, obtain basophilic granulocyte and other quantity of leucocyte.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104698157A (en) * | 2015-02-13 | 2015-06-10 | 中山市创艺生化工程有限公司 | Agent for blood cell analyzer |
| CN107735687A (en) * | 2015-06-30 | 2018-02-23 | 日本光电工业株式会社 | The method and apparatus for analyzing blood |
| CN107991151A (en) * | 2017-11-22 | 2018-05-04 | 中山市创艺生化工程有限公司 | A kind of reagent for hemocyte analyzers |
| CN108169468A (en) * | 2017-12-12 | 2018-06-15 | 山东兰桥医学科技有限公司 | A kind of dilution and its configuration method suitable for a variety of blood analysers |
| CN108489776A (en) * | 2018-02-07 | 2018-09-04 | 桂林优利特医疗电子有限公司 | A kind of optics Quality Control object and preparation method thereof |
| CN110132899A (en) * | 2018-02-02 | 2019-08-16 | 深圳市帝迈生物技术有限公司 | A kind of hemolytic agent for leukocyte differential count |
| CN110132908A (en) * | 2018-02-09 | 2019-08-16 | 深圳市帝迈生物技术有限公司 | A kind of cell detection kit and its application |
| CN110542640A (en) * | 2019-08-12 | 2019-12-06 | 江苏美诚生物科技有限公司 | Leukocyte classification kit for five-classification blood cell analyzer |
| CN111849736A (en) * | 2019-04-25 | 2020-10-30 | 深圳市帝迈生物技术有限公司 | Animal blood cell measuring method and animal blood analyzing apparatus |
| CN112014196A (en) * | 2020-08-15 | 2020-12-01 | 湖南伊鸿健康科技有限公司 | Dilution method for classifying and counting white blood cells |
| CN114047109A (en) * | 2022-01-11 | 2022-02-15 | 深圳市帝迈生物技术有限公司 | Sample analyzer and counting method thereof |
| CN114127535A (en) * | 2019-06-19 | 2022-03-01 | 深圳迈瑞动物医疗科技有限公司 | Cell analyzer, method for classifying leukocytes based on impedance method and computer-readable storage medium |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104698157A (en) * | 2015-02-13 | 2015-06-10 | 中山市创艺生化工程有限公司 | Agent for blood cell analyzer |
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| CN107735687A (en) * | 2015-06-30 | 2018-02-23 | 日本光电工业株式会社 | The method and apparatus for analyzing blood |
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| CN108169468A (en) * | 2017-12-12 | 2018-06-15 | 山东兰桥医学科技有限公司 | A kind of dilution and its configuration method suitable for a variety of blood analysers |
| CN110132899A (en) * | 2018-02-02 | 2019-08-16 | 深圳市帝迈生物技术有限公司 | A kind of hemolytic agent for leukocyte differential count |
| CN108489776A (en) * | 2018-02-07 | 2018-09-04 | 桂林优利特医疗电子有限公司 | A kind of optics Quality Control object and preparation method thereof |
| CN110132908A (en) * | 2018-02-09 | 2019-08-16 | 深圳市帝迈生物技术有限公司 | A kind of cell detection kit and its application |
| CN110132908B (en) * | 2018-02-09 | 2023-09-15 | 深圳市帝迈生物技术有限公司 | Cell detection kit and application thereof |
| CN111849736A (en) * | 2019-04-25 | 2020-10-30 | 深圳市帝迈生物技术有限公司 | Animal blood cell measuring method and animal blood analyzing apparatus |
| CN114127535A (en) * | 2019-06-19 | 2022-03-01 | 深圳迈瑞动物医疗科技有限公司 | Cell analyzer, method for classifying leukocytes based on impedance method and computer-readable storage medium |
| CN110542640A (en) * | 2019-08-12 | 2019-12-06 | 江苏美诚生物科技有限公司 | Leukocyte classification kit for five-classification blood cell analyzer |
| CN112014196A (en) * | 2020-08-15 | 2020-12-01 | 湖南伊鸿健康科技有限公司 | Dilution method for classifying and counting white blood cells |
| CN114047109A (en) * | 2022-01-11 | 2022-02-15 | 深圳市帝迈生物技术有限公司 | Sample analyzer and counting method thereof |
| CN114047109B (en) * | 2022-01-11 | 2022-06-21 | 深圳市帝迈生物技术有限公司 | Sample analyzer and counting method thereof |
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