CN106885896A - A kind of new hemolytic agent and preparation method thereof - Google Patents
A kind of new hemolytic agent and preparation method thereof Download PDFInfo
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- CN106885896A CN106885896A CN201710199956.2A CN201710199956A CN106885896A CN 106885896 A CN106885896 A CN 106885896A CN 201710199956 A CN201710199956 A CN 201710199956A CN 106885896 A CN106885896 A CN 106885896A
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- hemolytic agent
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- 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
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
Abstract
The present invention relates to a kind of new hemolytic agent, it is characterized in that containing certain density thymol, and the preparation method of the hemolytic agent is disclosed.The new hemolytic agent for preparing in the method, reliable and stable, solubility is good.Inventor is experimentally confirmed after using the new hemolytic agent to whole blood and erythrocyte hemolysis; more lipid compositions are detected in follow-up Analysis of blood lipid; it is able to demonstrate that it being capable of lipid component in protection system to greatest extent; reduce lipid degradation and destruction; and then external mammalian whole blood and erythrocytolysis are applied to, in the clinical examination such as blood and serum analysis field for clinical examination provides facility.Thymol whole content in new hemolytic agent can be 0.001%~0.2% (concentration), and this kind of purposes of thymol, it is expected to improve the accuracy of clinical lipid inspection and analysis, reduce inspection error.
Description
Technical field
The present invention relates to a kind of new hemolytic agent, it is characterized in that contain certain density thymol, and it is molten to disclose this
The preparation method of blood agent.The new hemolytic agent for preparing in the method, reliable and stable, solubility is good, new using this
After hemolytic agent dissolving whole blood, lipid component can be to greatest extent protected, for follow-up Analysis of blood lipid provides facility.The hemolytic agent can be with
For external mammalian whole blood and erythrocytolysis, the clinical examination such as blood and serum analysis field is applied to.
Background technology
Blood donor's a large amount of edible fat based foods before blood sampling, after being absorbed by the body, main component can be formed in blood is
The chylomicron of triglycerides, cholesterol, causes chylemia.Serious chylemia not only influences blood quality, especially freezes
The quality of blood plasma, and be difficult for clinic received, often result in the waste of blood.Accordingly, it would be desirable in the very first time of blood collecting and supplying
Judge the lipid chyle content of blood donor.Additionally, blood and plasma triglyceride, Determination of Cholesterol Content are also clinical laboratory
Routine inspection project, and during blood and plasma treatment, if lipid and lipid chyle surprisingly degrade or destroy, Shi Biying
The accuracy of inspection result is rung, mistake is brought to blood collecting and supplying and clinical examination.Therefore, should during blood and plasma treatment
The destruction of lipid is avoided as far as possible, and can protect lipid and the hemolytic agent of chyle to have prospect greatly.
Hemolytic agent conventional at present typically contains acid ingredient, based on acetic acid or propionic acid, in rapid damage red blood cell
Meanwhile, different degrees of irreversible destruction is caused to the lipid component in blood.If having control group, phase in subsequent detection
Can be made up by control with the lipid destruction of degree, but in quantitative determination, such as blood collecting and supplying moment, blood donor's blood
Lipid absolute value in liquid is most important, is directly connected to the home to return to of blood sample, it is therefore desirable to protect lipid to greatest extent,
With ensure that lipid and chyle detect it is accurate with it is credible.
Thymol also known as Thymol, are China《Food additives use sanitary standard》Regulation allows the food for using
Spices, is mainly used in preparing the essence such as cough syrup, American mint, chewing gum and spice.Additionally, the sterilized work of thymol
With stronger than phenol, and toxicity is low, there is sterilized, fungicidal action to oral cavity mucosa, has anti-corrosion, local anesthetic action to carious tooth chamber,
For oral cavity, the disinfection of throat, dermatophytosis, actinomycosis and otitis.Tra cheo-oesophageal voice can be promoted, be conducive to gas
The secretion of pipe mucus, easily plays phlegm-dispelling functions, then added with bactericidal action, therefore can be used to treat tracheitis, pertussis etc., can 5~
The effect of 100% kill protoscolex is reached in 10min, effect is rapid reliable, small toxicity.
In recent years studies have found that, thymol can reduce degradation of lipid with contaminated with lipid, therefore add in hemolytic agent
Plus a certain proportion of thymol, it is possible to the loss of lipid is reduced, and then increases the accuracy of Analysis of blood lipid.
Therefore the invention discloses a kind of new haemolysis agent prescription and preparation method thereof, add by existing hemolytic agent
Plus adapt to the thymol of concentration.Process same whole blood and red blood cell using the hemolytic agent, as a result be not added with thymol
Basic hemolytic agent compares, it is found that the blood lipid level detected after the hemolytic agent haemolysis is higher than basic hemolytic agent, it was demonstrated that this new haemolysis
Agent can to greatest extent protect lipid component, for follow-up Analysis of blood lipid provides facility.
The content of the invention
The present invention relates to a kind of new hemolytic agent, it is characterized in that contain certain density thymol (Thymol), and it is public
The preparation method of the hemolytic agent has been opened, the new hemolytic agent for preparing in the method, reliable and stable, solubility is good.Hair
A person of good sense has been experimentally confirmed the thymol that suitable concentration is added in hemolytic agent, can obtain new hemolytic agent.Using this
After new hemolytic agent is to whole blood and erythrocyte hemolysis, lipid component can be to greatest extent protected, for follow-up Analysis of blood lipid is provided just
Profit.The hemolytic agent can be used for external mammalian whole blood and erythrocytolysis, be applied to the clinical inspection such as blood and serum analysis
Test field.
The new hemolytic agent includes thymol and other basic hemolytic agent compositions.As the thyme of main ingredient in the present invention
Phenol is generally sterling, or semifinished product or drug extract containing thymol.Thymol is whole in new hemolytic agent
Content can be 0.001%~0.2% (concentration), preferably 0.01%~0.1% (concentration), more preferably 0.02%~
0.09% (concentration).
Preparation method
Material
Thymol, CAS numberings：89-83-8, molecular weight 150.22, white crystals or crystalline powder
Basic hemolytic agent：Can be with the hemolytic agent of commodity in use, it is also possible to voluntarily prepare.The hemolytic agent second commonly used at present
Acid-ethanol group, formaldehyde-acetic acid group, isopropanol-ethanol group etc..
The preparation of the new hemolytic agent containing thymol：A certain amount of thymol is added in basic hemolytic agent, control is eventually
Concentration, it is standby in 4 DEG C of refrigerators using again with 0.22 μm of millipore filter filtration sterilization.Nutrient solution is washed using preceding 90ml to add
10ml umbilical seras, use after 37 DEG C of pre-temperatures.
Compliance test result
Identical whole blood sample or red blood cell are dissolved using new hemolytic agent and control hemolytic agent, i.e., according to haemolysis
Ratio mixes hemolytic agent and whole blood or red blood cell, and slight concussion, stands 5 minutes at room temperature, checks that lysate is uniformly saturating
Bright being dissolves complete.
By determining the lipid components of hemolytic system, including T-CHOL (TG) and total triglyceride (TC) content, evaluate
The lipid loss of hemolytic system, as a result found, according to containing that preparation method disclosed by the invention is obtained after addition thymol
The new hemolytic agent of thymol, reliable and stable, solubility is good, using the new hemolytic agent to whole blood or erythrocyte hemolysis
Afterwards, lipid components content is higher than basic hemolytic agent in hemolytic system, illustrates that new hemolytic agent plays the role of to protect lipid.
Conclusion
Hemolytic agent containing thymol involved in the present invention can very well keep system after whole blood and erythrocyte hemolysis
Interior lipid components, the accuracy for improving lipid analysis in subsequent analysis, if energy popularization and application, will there is preferably economic effect
Benefit and social benefit.
Embodiment one：
New hemolytic agent containing 2/0,000 thymols (0.02%) is prepared and hemolyzing effect analysis
1st, material：
(1) thymol, 89-83-8, Sigma-Aldrich Products, molecular weight 150.22, article No. T0501, room temperature
Preserve, the effect phase was on 09 11st, 2018.
(2) basic hemolytic agent：Using methanol-acetic acid group hemolytic agent.Methyl alcohol 10ml, acetic acid 6ml, bromohexadecane base front three
Amine 3g, plus physiological saline is to 1000ml, brown bottle is contained after dissolution filter, room temperature preservation.
(3) whole blood sample：The whole blood sample for using is provided for volunteer, from air force general hospital Physical Check-Ups.Whole blood
Product are injected directly into the special blood-drawing pipe containing anticoagulant heparin, 4 DEG C of preservations, prevent pollution and haemolysis.Note：Human blood belongs to
In potential source biomolecule dangerous goods, please disposed according to international organization and national relevant laws and regulations.
(4) blood fat kit：T-CHOL (TG) determines kit and triglyceride (TC) determines kit, Sigma-
Aldrich product, article No. is respectively MAK043-1KT and TR0100-1KT.
2nd, method：
(1) prepared by the new hemolytic agent containing 2/0,000 thymols (0.02%)：1.000g thymols accurately are weighed, is added
50ml physiological saline solutions, are made 2% working solution, then the working solution of 1ml 2% is taken with pipettor, and addition 99ml's is basic molten
Shaken up in blood agent.
(2) haemolysis：By hemolytic agent (basic hemolytic agent and new hemolytic agent) containing thymol with fresh whole blood according to 20:
1 ratio mixing, slight concussion, stands 5 minutes at room temperature, checks that uniform bright being of lysate dissolves complete.
(3) lipid determination：Kit is determined using T-CHOL and triglyceride determines kit, according to kit method
Operation.
(4) statistical analysis：Using all measurement data measured values of the version softwares of SAS 8.01 result using mean ±
Standard deviationRepresent, all index determining values carry out selection check method after test of normality, multigroup is compared using single
Analysis of variance is compared (compare between group using minimum significantly method i.e. LSD inspections) two-by-two, is compared between two groups and is used Dan Yin
The conspicuousness of plain variance analysis test difference, statistics is that difference is statistically significant with P ＜ 0.05.
3rd, result：
Hemolytic system does not see difference from naked eyes after two kinds of hemolytic agent haemolysis.
Analysis of blood lipid result is shown in Fig. 2.
As can be seen that hemolytic system does not see difference from naked eyes after two kinds of hemolytic agent haemolysis, but lipid determination result shows,
After hemolytic agent haemolysis containing 2/10000ths thymols, blood lipid level is higher than the basic hemolytic agent without thymol, although
There is no difference between group, but one-to-one difference is positive number, the P compared with 0<0.01, illustrate the molten of 2/10000ths thymols
After blood agent is to each whole blood sample haemolysis, T-CHOL and triglyceride blood lipid level are above without thymol in system
Basic hemolytic agent.
4th, conclusion
The above results prove that the hemolytic agent containing 0.02% thymol has to blood lipid level in system after whole blood haemolysis
Protective effect, the hemolytic agent containing thymol can be used for the whole blood haemolysis of lipid determination.
Embodiment two：
New hemolytic agent containing 9/0,000 thymols (0.09%) is prepared and hemolyzing effect analysis
1st, material：
(1) thymol, 89-83-8, Sigma-Aldrich Products, molecular weight 150.22, article No. T0501, room temperature
Preserve, the effect phase was on 09 11st, 2018.
(2) basic hemolytic agent：Using methanol-acetic acid group hemolytic agent.Methyl alcohol 10ml, acetic acid 6ml, bromohexadecane base front three
Amine 3g, plus physiological saline is to 1000ml, brown bottle is contained after dissolution filter, room temperature preservation.
(3) whole blood sample：The whole blood sample for using is provided for volunteer, from air force general hospital Physical Check-Ups.Whole blood
Product are injected directly into the special blood-drawing pipe containing anticoagulant heparin, 4 DEG C of preservations, prevent pollution and haemolysis.Note：Human blood belongs to
In potential source biomolecule dangerous goods, please disposed according to international organization and national relevant laws and regulations.
(4) blood fat kit：T-CHOL (TG) determines kit and triglyceride (TC) determines kit, Sigma-
Aldrich product, article No. is respectively MAK043-1KT and TR0100-1KT.
2nd, method：
(1) prepared by the new hemolytic agent containing 9/0,000 thymols (0.09%)：9.000g thymols accurately are weighed, is added
50ml physiological saline solutions, are made 9% working solution, then the working solution of 1ml 2% is taken with pipettor, and addition 99ml's is basic molten
Shaken up in blood agent.
(2) haemolysis：By hemolytic agent (basic hemolytic agent and new hemolytic agent) containing thymol with fresh whole blood according to 20:
1 ratio mixing, slight concussion, stands 5 minutes at room temperature, checks that uniform bright being of lysate dissolves complete.
(3) lipid determination：Kit is determined using T-CHOL and triglyceride determines kit, according to kit method
Operation.
(4) statistical analysis：Using all measurement data measured values of the version softwares of SAS 8.01 result using mean ±
Standard deviationRepresent, all index determining values carry out selection check method after test of normality, multigroup is compared using single
Analysis of variance is compared (compare between group using minimum significantly method i.e. LSD inspections) two-by-two, is compared between two groups and is used Dan Yin
The conspicuousness of plain variance analysis test difference, statistics is that difference is statistically significant with P ＜ 0.05.
3rd, result：
Hemolytic system does not see difference from naked eyes after two kinds of hemolytic agent haemolysis.Such as Fig. 1.
Analysis of blood lipid result is shown in Fig. 3.
As can be seen that hemolytic system does not see difference from naked eyes after two kinds of hemolytic agent haemolysis, but lipid determination result shows,
After hemolytic agent haemolysis containing 9/10000ths thymols, blood lipid level is higher than the basic hemolytic agent without thymol, although
There is no difference between group, but one-to-one difference is positive number, the P compared with 0<0.01, illustrate the molten of 9/10000ths thymols
After blood agent is to each red blood cell sample haemolysis, T-CHOL and triglyceride blood lipid level are above without thyme in system
The basic hemolytic agent of phenol.
4th, conclusion
The above results prove that the hemolytic agent containing 0.09% thymol has to blood lipid level in system after whole blood haemolysis
Protective effect, the hemolytic agent containing thymol can be used for the whole blood haemolysis of lipid determination.
Embodiment three：
New hemolytic agent containing 9/0,000 thymols (0.09%) is prepared and hemolyzing effect analysis
1st, material：
(1) thymol, 89-83-8, Sigma-Aldrich Products, molecular weight 150.22, article No. T0501, room temperature
Preserve, the effect phase was on 09 11st, 2018.
(2) basic hemolytic agent：Using methanol-acetic acid group hemolytic agent.Methyl alcohol 10ml, acetic acid 6ml, bromohexadecane base front three
Amine 3g, plus physiological saline is to 1000ml, brown bottle is contained after dissolution filter, room temperature preservation.
(3) red blood cell sample：The whole blood sample of use is separated and obtained, and whole blood sample is volunteer from health, by sky
Army's hospital general's Physical Check-Ups is obtained.Whole blood sample is injected directly into the special blood-drawing pipe containing anticoagulant heparin, under the conditions of 4 DEG C
3000g is centrifuged 15min, removes plasma supernatant, adds the physiological saline of same volume, gently mixes, 4 DEG C of preservations, prevents pollution.Note
Meaning：Human blood belongs to potential source biomolecule dangerous goods, is please disposed according to international organization and national relevant laws and regulations.
(4) blood fat kit：T-CHOL (TG) determines kit and triglyceride (TC) determines kit, Sigma-
Aldrich product, article No. is respectively MAK043-1KT and TR0100-1KT.
2nd, method：
(1) prepared by the new hemolytic agent containing 9/0,000 thymols (0.09%)：9.000g thymols accurately are weighed, is added
50ml physiological saline solutions, are made 9% working solution, then the working solution of 1ml 2% is taken with pipettor, and addition 99ml's is basic molten
Shaken up in blood agent.
(2) haemolysis：By hemolytic agent (basic hemolytic agent and new hemolytic agent) containing thymol and fresh red blood cell preparation
According to 20:The ratio of 1 (volume ratio) mixes, and slight concussion, stands 5 minutes at room temperature, checks that lysate is uniformly bright and is
Dissolving is complete.
(3) lipid determination：Kit is determined using T-CHOL and triglyceride determines kit, according to kit method
Operation.
(4) statistical analysis：Using all measurement data measured values of the version softwares of SAS 8.01 result using mean ±
Standard deviationRepresent, all index determining values carry out selection check method after test of normality, multigroup is compared using single
Analysis of variance is compared (compare between group using minimum significantly method i.e. LSD inspections) two-by-two, is compared between two groups and is used Dan Yin
The conspicuousness of plain variance analysis test difference, statistics is that difference is statistically significant with P ＜ 0.05.
3rd, result：
Hemolytic system does not see difference, such as Fig. 1 from naked eyes after two kinds of hemolytic agent haemolysis.
Analysis of blood lipid result is shown in Fig. 4.
As can be seen that hemolytic system does not see difference from naked eyes after two kinds of hemolytic agent haemolysis, but lipid determination result shows,
After hemolytic agent containing 9/10000ths thymols is to erythrocyte hemolysis, blood lipid level is higher than the basic haemolysis without thymol
Agent, although not having difference between group, one-to-one difference is positive number, the P compared with 0<0.01, illustrate 9/10000ths thymes
After the hemolytic agent of phenol is to each red blood cell sample haemolysis, T-CHOL and triglyceride blood lipid level are above being free of in system
The basic hemolytic agent of thymol.
4th, conclusion
The above results prove that blood lipid level has and protects in the hemolytic agent erythrocyte hemolysis system containing 0.09% thymol
Shield is acted on, and the hemolytic agent containing thymol can be used for the erythrocyte hemolysis of lipid determination.
Brief description of the drawings：
Fig. 1 hemolytic agent hemolyzing effects, A：Basic hemolytic agent haemolysis result without thymol；B：Containing 2/0,000 thymes
The new hemolytic agent haemolysis result of phenol；C：New hemolytic agent haemolysis result containing 9/0,000 thymols；D：Containing 9/0,000 thymols
New hemolytic agent to erythrocyte hemolysis result.
Fig. 2：Containing 0.02% thymol hemolytic agent to measuring lipids contents, * * after whole blood haemolysis:P<0.01, it is molten with basis
Blood agent group compares, n=24
Fig. 3：Containing 0.09% thymol hemolytic agent to measuring lipids contents, * * after whole blood haemolysis:P<0.01, it is molten with basis
Blood agent group compares, n=25
Fig. 4：Contain to measuring T-CHOL and total triglyceride after erythrocyte hemolysis containing 0.09% thymol hemolytic agent
Amount, * *:P<0.01, compare with basic hemolytic agent group, n=25.
Claims (5)
1. a kind of hemolytic agent containing thymol, it is characterized in that the thymol containing 0.001%~0.2% (concentration), can
For whole blood and erythrocyte hemolysis.
2. application of the thymol separately as medicine in terms of hemolytic agent.
3. application of the thymol as additive in terms of hemolytic agent.
4. hemolytic agent as claimed in claim 1, its formulation can be liquid, or instant dry powder.
5., according to claim 1, the concentration range lower limit of the application described in 2,3 and 4, wherein thymol is 0.001%, the upper limit
It is 0.2%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710199956.2A CN106885896A (en) | 2017-03-30 | 2017-03-30 | A kind of new hemolytic agent and preparation method thereof |
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| CN201710199956.2A CN106885896A (en) | 2017-03-30 | 2017-03-30 | A kind of new hemolytic agent and preparation method thereof |
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| CN106885896A true CN106885896A (en) | 2017-06-23 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2017
- 2017-03-30 CN CN201710199956.2A patent/CN106885896A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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