CN112162103A - Full-automatic thromboelastogram activated blood coagulation detection reagent and preparation method and application thereof - Google Patents
Full-automatic thromboelastogram activated blood coagulation detection reagent and preparation method and application thereof Download PDFInfo
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- 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
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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Abstract
The invention discloses a full-automatic thromboelastogram activated blood coagulation detection reagent and a preparation method and application thereof, wherein the reagent comprises the following components: buffer, activator of intrinsic coagulation and phospholipid; in the reagent, the concentration of the endogenous blood coagulation activator is 0.1-10 g/L, and the concentration of the phospholipid is 0.1-5 g/L. The reagent has simple components and low preparation cost, is used for detecting the blood coagulation function by the full-automatic thromboelastogram under the specific proportion, can more truly represent the states of blood coagulation and fibrinolysis in the body of a patient, and has the advantages of high detection efficiency and good stability.
Description
Technical Field
The invention relates to the technical field of blood coagulation, and particularly relates to a full-automatic thromboelastogram activated blood coagulation detection reagent and a preparation method and application thereof.
Background
Clinically, the evaluation of human blood coagulation function receives more and more attention, and the probability that hemorrhage appears in the art, postoperative thrombus takes place can be forecasted in the evaluation of preceding art and postoperative blood coagulation to the clinician can prepare in advance, avoids taking place unfortunate incident. Currently, the clinical routine coagulation screening includes Prothrombin Time (PT), Thrombin Time (TT), Activated Partial Thrombin Time (APTT), Fibrinogen (FIB), etc., and these routine items subjectively separate the complex coagulation cascade in vivo. However, the conventional blood coagulation item test usually adopts blood plasma, the participation of visible components in the blood is lost, and the measured result cannot truly represent the blood coagulation state in a patient body.
Thromboelastography (TEG) was invented by Harter, german in 1948. The blood transfusion system has good effect when being widely used for blood transfusion in clinical guidance operation in the 1980 s, and is the most important index for monitoring the blood coagulation function in the perioperative period at present. TEG monitors the physical properties of blood clots based on the following principles: a specially prepared cylindrical cup, still containing blood, was rotated at an angle of 4 ° 45' with each rotation lasting 10 seconds. The movement of the blood sample is monitored by a needle suspended by a spiral wire and immersed in the blood sample. After the fibrin platelet complex adheres the cup and the needle together, rotational force generated by the rotation of the cup can be transmitted to the needle in the blood sample. The strength of the fibrin-platelet complex can affect the amplitude of the needle movement so that a strong blood clot can synchronize the needle movement with the cup movement. Thus, the amplitude of the needle movement is directly related to the strength of the blood clot that has formed. When the clot is retracted or dissolved, the needle is decoupled from the clot and the motion of the cup is no longer transmitted to the needle. The rotation of the needle is converted by an electromechanical sensor into an electronic signal that can be monitored by a computer. The TEG thromboelastography device can output blood coagulation parameters such as blood coagulation time, blood coagulation rate and blood clot strength, and the whole process of fibrinolysis (see fig. 1).
The existing TEG full-automatic thromboelastography activated blood coagulation detection reagent has some problems, such as high cost, poor detection stability and the like.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a full-automatic thromboelastogram activated blood coagulation detection reagent and a preparation method and application thereof.
The invention is realized by the following steps:
in a first aspect, embodiments provide a full-automatic thromboelastogram activated coagulation detection reagent, which comprises the following components: buffer, activator of intrinsic coagulation and phospholipid;
in the reagent, the concentration of the endogenous blood coagulation activator is 0.1-10 g/L, and the concentration of the phospholipid is 0.1-5 g/L.
In a second aspect, embodiments provide a method of preparing a fully automated thromboelastography activated coagulation test reagent of the preceding embodiments, comprising mixing the components of the reagent.
In a third aspect, the embodiment provides the application of the fully automatic thromboelastography activated coagulation detection reagent described in the previous embodiment in preparing a fully automatic thromboelastography activated coagulation detection kit.
In a fourth aspect, embodiments provide a fully automatic thromboelastogram activated coagulation detection kit, which includes the fully automatic thromboelastogram activated coagulation detection reagent described in the previous embodiments.
In a fifth aspect, embodiments provide a method for detecting a coagulation function of a sample, which includes detecting the sample by using the fully automatic thromboelastography activated coagulation detection reagent described in the previous embodiments;
the methods are not aimed at the diagnosis or treatment of disease.
The invention has the following beneficial effects:
the embodiment of the invention provides a full-automatic thromboelastogram activated blood coagulation detection reagent and a preparation method and application thereof, wherein the detection reagent comprises the following components: buffer, activator of intrinsic coagulation and phospholipid; in the reagent, the concentration of the endogenous blood coagulation activator is 0.1-10 g/L, and the concentration of the phospholipid is 0.1-5 g/L. The reagent has simple components and low preparation cost, is used for detecting the blood coagulation function by the full-automatic thromboelastogram under the specific proportion, can more truly represent the states of blood coagulation and fibrinolysis in the body of a patient, and has the advantages of high detection efficiency and good stability.
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, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a diagram illustrating the elasticity of a thrombus in the background art;
FIG. 2 is the results of comparison of the R-values of the test sample 1 with the inlet sample of the control sample in the validation example 1;
FIG. 3 shows the comparison of K values of the test reagent of example 1 with the inlet reagent of the control example in the validation example 1;
FIG. 4 is a graph showing the comparison result of the Angle value of the reagent of example 1 in the validation example 1 with the inlet reagent of the control example;
FIG. 5 shows the comparison of MA values of the test reagent of example 1 and the inlet reagent of the control example in the validation example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment provides a full-automatic thromboelastogram activated blood coagulation detection reagent, which is applied to detection of a full-automatic thromboelastogram instrument and comprises the following components: buffer, activator of intrinsic coagulation and phospholipid;
in the detection reagent, the concentration of the endogenous blood coagulation activator is 0.1-10 g/L, and the concentration of the phospholipid is 0.1-5 g/L.
The inventor researches and discovers that the reagent for detecting the blood coagulation in the full-automatic thromboelastogram can more truly represent the coagulation and fibrinolysis states in the body of a patient and can stably and effectively carry out detection under the specific proportion. The concentrations of the intrinsic coagulation activator and the phospholipid are the concentrations when they are mixed in a buffer solution.
Specifically, an activator of intrinsic coagulation refers to an agent that, when added, initiates the pathway of intrinsic coagulation activation. The endogenous blood coagulation activator may have a concentration of 0.1g/L, 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L, 1.2g/L, 1.4g/L, 1.6g/L, 1.8g/L, 2.0g/L, 2.2g/L, 2.4g/L, 2.6g/L, 2.8g/L, 3.0g/L, 3.2g/L, 3.4g/L, 3.6g/L, 3.8g/L, 4.0g/L, 4.2g/L, 4.4g/L, 4.6g/L, 4.8g/L, 5.0g/L, 5.2g/L, 5.4g/L, 5.6g/L, 5.8g/L, 6.0g/L, 6.6g/L, 6g/L, 7.8g/L, 6g/L, 6.6g/L, 6g/, 7.2g/L, 7.4g/L, 7.6g/L, 7.8g/L, 8.0g/L, 8.2g/L, 8.4g/L, 8.6g/L, 8.8g/L, 9.0g/L, 9.2g/L, 9.4g/L, 9.6g/L, 9.8g/L, or 10.0 g/L.
The concentration of the phospholipid may be 0.1g/L, 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L, 1.2g/L, 1.4g/L, 1.6g/L, 1.8g/L, 2.0g/L, 2.2g/L, 2.4g/L, 2.6g/L, 2.8g/L, 3.0g/L, 3.2g/L, 3.4g/L, 3.6g/L, 3.8g/L, 4.0g/L, 4.2g/L, 4.4g/L, 4.6g/L, 4.8g/L, or 5.0 g/L.
Preferably, the concentration of the endogenous blood coagulation activator is 0.1-5 g/L, and the concentration of the phospholipid is 0.1-3 g/L; preferably, the concentration of the endogenous blood coagulation activator is 0.5-3 g/L, and the concentration of the mixed phospholipid is 0.5-1.5 g/L.
Preferably, the phospholipid is selected from at least one of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and phosphatidylglycerol; preferably, the phospholipid comprises at least two of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and phosphatidylglycerol; preferably, the activator of intrinsic coagulation is selected from the group consisting of: at least one of kaolin, ellagic acid, diatomaceous earth and kaolin.
In an alternative embodiment, the agent further comprises a preservative.
Preferably, the preservative is 0.1-2% by volume of the reagent. Specifically, the preservative may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% by volume.
Preferably, the volume percentage of the preservative is 0.1-0.5%. Within this range, the stability of the reagent is better.
Preferably, the preservative is selected from: at least one of proclin 300, sodium azide, gentamicin sulfate and thimerosal.
Preferably, the concentration of the buffer solution is 5-50 mM, and the pH value is 7.0-7.5. Specifically, the buffer may have a concentration of 5mM, 6mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, 22mM, 24mM, 26mM, 28mM, 30mM, 32mM, 34mM, 36mM, 38mM, 40mM, 42mM, 44mM, 46mM, 48mM, or 50 mM. The pH may be 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.
Preferably, the concentration of the buffer solution is 10-50 mM, and the pH value is 7.2-7.5. The stability of the reagent is better in this concentration range and pH range of the buffer.
In alternative embodiments, the buffer component in the buffer is selected from: any one of HEPES, imidazole, PBS, tris and barbital.
The embodiment of the invention provides a preparation method of a full-automatic thromboelastogram activated blood coagulation detection reagent, which comprises mixing the components of the reagent.
Specifically, the method comprises adding components (e.g., an activator of intrinsic coagulation, a phospholipid, and/or a preservative) to a buffer to bring the activator of intrinsic coagulation and the phospholipid to the concentrations described in any of the embodiments above.
The embodiment of the invention provides an application of the full-automatic thromboelastogram activated coagulation detection reagent in preparation of a full-automatic thromboelastogram activated coagulation detection kit.
The embodiment of the invention provides a full-automatic thromboelastogram activated blood coagulation detection kit, which is applied to detection of a full-automatic thromboelastogram instrument and comprises the full-automatic thromboelastogram activated blood coagulation detection reagent in any one of the embodiments.
In some embodiments, the kit further comprises a detection vessel.
In addition, the invention also provides a method for detecting the blood coagulation function of a sample, which is applied to the detection of a full-automatic thromboelastography instrument and comprises the steps of detecting the sample by using the full-automatic thromboelastography activated blood coagulation detection reagent according to any one of the embodiments;
the methods are not aimed at the diagnosis or treatment of disease.
The detection method of the fully automatic thromboelastogram apparatus is not particularly limited, and any detection method of the fully automatic thromboelastogram apparatus disclosed in the related art (or reference to the operation description of the fully automatic thromboelastogram apparatus) may be adopted, and the reagent described in any of the above embodiments is used, and the reagent falls within the scope of the present application.
It should be noted that, the method is not intended for diagnosis or treatment of diseases, and only performs the detection process, and does not interpret the result of the sample, for example, a third party detects the process of detecting the sample by using the reagent provided in the examples.
In some embodiments, the sample is a blood sample.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The invention provides a full-automatic thromboelastogram activated blood coagulation detection reagent, which comprises the following components: buffer solution, endogenous coagulation activator, phospholipid and preservative.
Wherein the endogenous coagulation activator is kaolin, and the concentration of the kaolin is 1 g/L; the concentration of the phospholipid is 1 g/L; the preservative is sodium azide, and the volume percentage is 0.1%; the buffer solution is tris buffer solution with a concentration of 40mM and a pH value of 7.4.
Example 2
The invention provides a full-automatic thromboelastogram activated blood coagulation detection reagent, which is approximately the same as the reagent provided in example 1, and is different only in the concentration of kaolin: the concentration of the kaolin is 0.5 g/L.
Example 3
The invention provides a full-automatic thromboelastogram activated blood coagulation detection reagent, which is approximately the same as the reagent provided in example 1, and is different only in the concentration of kaolin: the concentration of the kaolin is 1.5 g/L.
Example 4
The invention provides a full-automatic thromboelastogram activated blood coagulation detection reagent, which is approximately the same as the reagent provided in example 1, and is only different from the reagent provided in example 1 in the concentration of mixed phospholipid: the concentration of the mixed phospholipid is 0.5 g/L.
Example 5
The invention provides a full-automatic thromboelastogram activated blood coagulation detection reagent, which is approximately the same as the reagent provided in example 1, and is only different from the reagent provided in example 1 in the concentration of mixed phospholipid: the concentration of the mixed phospholipid is 1.5 g/L.
Example 6
The invention provides a full-automatic thromboelastogram activated blood coagulation detection reagent, which is approximately the same as the reagent provided in example 1, and is only different from the reagent provided in example 1 in the pH value of a buffer solution: the pH was 7.2.
Example 7
The invention provides a full-automatic thromboelastogram activated blood coagulation detection reagent, which is approximately the same as the reagent provided in example 1, and is different from the preservative: the preservative is gentamicin sulfate.
Verification example 1
The test effect of the fully automatic thromboelastogram activated blood coagulation test reagent provided in example 1 was verified.
The reagent provided in example 1 was applied to a fully automatic thromboelastogram apparatus to detect the blood coagulation function of a sample, and a set of control examples were provided in which R-, K-, Angle-and Ma-values were detected using a test reagent (TEG reagent).
Specifically, the R value refers to the time required from the start of the test to the formation of the first fibrin (amplitude of 2mm), reflecting the overall condition of the coagulation factors.
The K value is the time from the end of the R time to the time required for the amplitude to reach 20mm, reflecting the result of the combined effect of fibrin and platelets at the beginning of clotting, i.e. the rate of clot formation.
The Angle reflects the result of the interaction of fibrin and platelets at the onset of clotting. The included Angle between the tangent and the horizontal line is drawn from the clot formation point to the maximum curve radian of the elastogram, and the Angle is closely related to the K value and reflects the rate of clot formation.
The MA value is the maximum amplitude on the elastogram, reflecting the function of fibrinogen and platelets. The role of platelets is about 80% of the total, and platelet function includes quality and quantity, both of which affect the MA value.
And the results of the tests using the reagents provided in the control and example 1 were compared using the chi-square test and symmetry metrics.
Results of the experiment
For comparison of the R value, K value, Angle value and Ma value of example 1 and the comparative example, refer to FIGS. 2-5.
The results of the chi-square test are shown in table 1, and the results of the symmetry measure are shown in table 2.
TABLE 1 chi fang test
Remarking: in table 1, the expected count for the a.1 cell (25%) is less than 5, with a minimum expected count of 3.93; b. only for 22 table calculations; c. a binomial distribution is used.
TABLE 2 symmetry metric
Value of | Progressive standard errora | Approximation Tb | An approximation Sig. | ||
Scaling by scalar quantity | Coefficient of dependence | .680 | .000 | ||
By interval | R of Pearson | .927 | .069 | 12.564 | .000c |
In sequence | Spearman correlation | .927 | .069 | 12.564 | .000c |
Consistency metric | Kappa | .924 | .074 | 4.903 | .000 |
N in effective case | 28 |
Remarking: a. no null hypothesis is assumed; b. the null hypothesis is assumed using progressive standard error.
As shown by the comparison of the methodology (chi-square test and symmetry measurement), the test results of the imported reagent of the control example and the reagent provided in example 1 are consistent, and the kappa value is equal to 0.924 > 0.8.
Verification example 2
The stability of the fully automated thromboelastogram activated coagulation assay reagent provided in example 1 was verified.
A plurality of groups of the reagents provided in example 1 and the imported reagent (same as the verification example 1) are applied to a full-automatic thromboelastography instrument at 37 ℃ for 18 days, and the blood agglutination function of clinical samples is detected within 18 days. The results are shown in Table 3.
TABLE 3 test results
From the results, it is clear that the stability of the reagent provided in example 1 is significantly superior to the stability of the prior art assay.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The full-automatic thromboelastogram activated blood coagulation detection reagent is applied to detection of a full-automatic thromboelastogram instrument, and is characterized by comprising the following components: buffer, activator of intrinsic coagulation and phospholipid;
in the detection reagent, the concentration of the endogenous blood coagulation activator is 0.1-10 g/L, and the concentration of the phospholipid is 0.1-5 g/L.
2. The full-automatic thromboelastogram activated blood coagulation detection reagent according to claim 1, wherein the concentration of the intrinsic blood coagulation activator is 0.1-5 g/L, and the concentration of the phospholipid is 0.1-3 g/L;
preferably, the concentration of the endogenous blood coagulation activator is 0.5-3 g/L, and the concentration of the phospholipid is 0.5-1.5 g/L.
3. The reagent for detecting full-automatic thromboelastogram-activated coagulation according to claim 1 or 2, wherein the phospholipid is selected from at least one of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and phosphatidylglycerol;
preferably, the phospholipid comprises at least two of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and phosphatidylglycerol;
preferably, the activator of intrinsic coagulation is selected from the group consisting of: at least one of kaolin, ellagic acid, diatomaceous earth and kaolin.
4. The fully automated thromboelastography activated coagulation assay reagent of claim 3, wherein the assay reagent further comprises a preservative;
preferably, in the detection reagent, the volume percentage of the preservative is 0.1-2%;
preferably, the volume percentage of the preservative is 0.1-0.5%;
preferably, the preservative is selected from: at least one of proclin 300, sodium azide, gentamicin sulfate and thimerosal.
5. The reagent for detecting full-automatic thromboelastogram activated coagulation according to claim 4, wherein the concentration of the buffer solution is 5-50 mM, and the pH value is 7.0-7.5;
preferably, the concentration of the buffer solution is 10-50 mM, and the pH value is 7.2-7.5.
6. The reagent of claim 5, wherein the buffer component of the buffer is selected from the group consisting of: any one of HEPES, imidazole, PBS, tris and barbital.
7. A method for preparing the fully automatic thromboelastography activated blood coagulation detection reagent as claimed in any one of claims 1 to 6, which comprises mixing the components of the detection reagent.
8. The application of the full-automatic thromboelastogram activated coagulation detection reagent as claimed in any one of claims 1-6 in preparation of a full-automatic thromboelastogram activated coagulation detection kit.
9. A full-automatic thromboelastogram activated blood coagulation detection kit, which is characterized by comprising the full-automatic thromboelastogram activated blood coagulation detection reagent as claimed in any one of claims 1-6.
10. A method for detecting the blood coagulation function of a sample, which is applied to the detection of a full-automatic thromboelastogram instrument and is characterized by comprising the steps of detecting the sample by using the full-automatic thromboelastogram activated blood coagulation detection reagent as claimed in any one of claims 1-6;
the methods are not aimed at the diagnosis or treatment of disease.
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