CN112730769A - Platelet aggregation functional adenosine diphosphate cup detection reagent and preparation method thereof - Google Patents
Platelet aggregation functional adenosine diphosphate cup detection reagent and preparation method thereof Download PDFInfo
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- G—PHYSICS
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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
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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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
- G01N33/86—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
Abstract
The invention belongs to the technical field of blood coagulation detection, and particularly relates to a platelet aggregation functional adenosine diphosphate detection reagent and a preparation method thereof. The adenosine diphosphate detection reagent comprises adenosine diphosphate disodium and a freeze-drying protective agent, wherein the freeze-drying protective agent comprises a saccharide freeze-drying protective agent and a polymer freeze-drying protective agent. The detection reagent fully considers different reactivities of the diagnostic reagent to antiplatelet drugs, and has better sensitivity to clopidogrel, ticagrelor, prasugrel and the like which are commonly used clinically at present. The adenosine diphosphate cup detection reagent is used for heparin sodium anticoagulated blood detection after medicine taking, and has the advantages of good detection result accuracy and high precision in batch and between batches.
Description
Technical Field
The invention belongs to the technical field of blood coagulation functions, and particularly relates to a platelet aggregation function detection kit and a preparation method thereof.
Background
The Thromboelastogram (TEG) is a blood coagulation function analyzer which carries out real-time, dynamic and continuous monitoring on the whole processes of platelet aggregation, blood coagulation, fibrinolysis and the like, and carries out qualitative or quantitative analysis on the blood coagulation condition of a patient.
The invention of the thromboelastogram instrument by Harter of German in 1948 is applied to guide the blood transfusion in clinical operation, and is one of the most important means for detecting the blood coagulation function at present. The principle of the thromboelastogram for detecting the blood coagulation function is that a sensor probe is immersed in a blood sample, a viscous force exists between the blood sample and the sensor probe, the viscous force changes along with changes of platelet aggregation, blood coagulation, fibrinolysis and the like, a suspension wire of a sensor generates torsional deformation under the action of the viscous force, the torsional deformation is detected by a detection unit and is converted into an electric signal to be fed back to a control system, the control system carries out operation after receiving the changed signal, and an operation result is displayed on a computer screen in the form of a curve graph and real-time data.
Thromboelastogram (TEG) uses anticoagulated whole blood to activate the coagulation and fibrinolysis system from the extrinsic coagulation pathway, thereby reflecting the whole process of human coagulation factors, platelets, fibrin and fibrinolysis more comprehensively and truly.
The main functions of platelets are coagulation, hemostasis, and repair of damaged blood vessels. Platelet surface coagulation function related receptor ADP receptor, alpha and beta-adrenoreceptor, thrombin receptor, collagen receptor (GP Ia/IIa, GP IV, GP-VI), TXA2/PGH2Receptor, PGI2/PGE1Receptors, 5-hydroxytryptamine receptors, heparin receptors, PFA (platelet activating factor) receptors, fibrinogen receptors, Ca2+Receptor fibronectin receptors, platelet factor 4 receptors, angiotensin II receptors, LDL receptors, HDL receptors, vWF receptors (GPIb/IX) and GPIIb/IIIa (fibrinogen receptors), and the like. The GP IIb/IIIa membrane receptor is an adhesive glycoprotein on the platelet surface, a most abundant integrin on the platelet surface, with a size of about 5X 104~8×104A GPIIb/IIIa membrane receptor. ADP, AA, thrombin, etc. can activate plateletThe conformation of the surface GP IIb/IIIa membrane receptor is changed, so that the affinity of the GP IIb/IIIa membrane receptor to soluble fibrinogen, vWF and the like is increased, and finally platelets are gathered on bridging fibrin and vWF to form platelet thrombus.
ADP is the most important platelet aggregation inducing substance in humans. In one aspect, ADP is bound to platelet membrane receptor P2Y1After the receptor is combined, the coupled protein Gq activates phospholipase C to enable Ca2+From the extracellular stream into the intracellular, intracellular Ca2+The increase in concentration activates protein kinase C, causing platelet deformation and aggregation; ADP and platelet P, on the other hand2Y12Membrane receptor binding, coupled with Gi protein, inhibits activation of adenylate cyclase AC, decreases cAMP levels in platelets, induces platelet aggregation. Clopidogrel by liver cytochrome P450The enzyme metabolizes to form the active substance, irreversibly binds to the platelets P2Y12The membrane receptor combination blocks the inhibition effect of ADP on adenylate cyclase AC, promotes the phosphorylation of the phosphoprotein VASP (dependent on cyclic adenosine monophosphate and stimulated by prostaglandin E1), and inhibits the activation of the GP IIb/IIIa membrane receptor, thereby inhibiting the platelet aggregation.
Clopidogrel is used as a common anti-platelet aggregation drug and is widely used for complications caused by acute coronary syndrome, ischemic cerebral thrombosis, arteriosclerosis, thromboembolism and the like. At present, the Thromboelastogram (TEG) lacks enough sensitivity in detecting the platelet aggregation function, namely, the TEG cannot be effectively detected from a patient sample taking a clopidogrel drug or can be effectively detected, but compared with other methods, the TEG cannot truly reflect the degree of the inhibition of platelets.
Disclosure of Invention
The invention provides a platelet aggregation functional adenosine diphosphate cup detection reagent, aiming at the defects and problems that the existing detection reagent can not effectively detect a patient sample taking clopidogrel drugs, and can not truly reflect the degree of platelet inhibition compared with other methods even if the existing detection reagent can effectively detect the patient sample taking clopidogrel drugs.
The technical scheme adopted by the invention for solving the technical problems is as follows: a reagent for detecting platelet aggregation functional adenosine diphosphate cup comprises disodium adenosine diphosphate and freeze-drying protective agent.
In the reagent for detecting the platelet aggregation functional adenosine diphosphate cup, the addition amount of the adenosine diphosphate is 0.012%.
The freeze-drying protective agent comprises a saccharide freeze-drying protective agent and a polymer freeze-drying protective agent.
In the reagent for detecting platelet aggregation functional adenosine diphosphate cup, the saccharide freeze-drying protective agent is one or more of trehalose, sucrose, glucan and glucose.
According to the reagent for detecting platelet aggregation functional adenosine diphosphate cup, the saccharide protective agent is trehalose, and the addition amount of the trehalose is 1%.
In the reagent for detecting platelet aggregation functional adenosine diphosphate cup, the polymer freeze-drying protective agent is one or more of bovine serum albumin, polyvinylpyrrolidone and polyethylene glycol.
According to the reagent for detecting platelet aggregation functional adenosine diphosphate cup, the polymer freeze-drying protective agent is bovine serum albumin, and the addition amount of the bovine serum albumin is 2%.
The invention also provides a preparation method of the platelet aggregation functional adenosine diphosphate cup detection reagent, which comprises the following steps:
(1) weighing the freeze-drying protective agent according to the formula amount, and adding distilled water or purified water to fully dissolve to obtain a freeze-drying protective agent solution;
(2) weighing disodium adenosine diphosphate according to the formula amount, pouring the disodium adenosine diphosphate into a freeze-drying protective agent solution, and uniformly mixing to obtain adenosine diphosphate detection reagent mother liquor;
(3) subpackaging the adenosine diphosphate detection reagent into penicillin bottles according to 20-60 microliter per bottle to obtain a finished product adenosine diphosphate detection reagent;
(4) and (3) carrying out vacuum freeze drying on the adenosine diphosphate detection reagent to obtain an adenosine diphosphate reagent freeze-dried product.
According to the preparation method of the adenosine diphosphate cup detection reagent with the platelet aggregation function, the adenosine diphosphate detection reagent is subpackaged into penicillin bottles according to 40 microliter per bottle.
The invention has the beneficial effects that: the invention fully considers the different reactivities of the diagnostic reagent to antiplatelet drugs, and has better sensitivity to clopidogrel, ticagrelor, prasugrel and the like which are commonly used clinically at present. The adenosine diphosphate cup detection reagent is used for heparin sodium anticoagulation blood detection after medicine taking, and has good detection result accuracy and high precision in batch and between batches.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: the embodiment provides a platelet aggregation functional adenosine diphosphate cup detection reagent, which comprises adenosine diphosphate disodium and a freeze-drying protective agent, wherein the freeze-drying protective agent comprises a saccharide freeze-drying protective agent and a polymer freeze-drying protective agent, and the saccharide freeze-drying protective agent can be any one or more of trehalose, sucrose, glucan and glucose; the polymer freeze-drying protective agent can be any one or more of bovine serum albumin, polyvinylpyrrolidone and polyethylene glycol. Trehalose and bovine serum albumin are selected as freeze-drying protective agents in the implementation.
The total amount of the adenosine diphosphate cup detection reagent in the embodiment is 40 microliters, and the dosage of each raw material according to the volume ratio is respectively as follows: disodium adenosine diphosphate 0.012%, bovine serum albumin 2.0%, trehalose 1.0%, the rest is distilled water.
The preparation method comprises the following steps:
(1) weighing bovine serum albumin and trehalose according to the formula amount, and adding distilled water or purified water to fully dissolve to obtain a freeze-drying protective agent solution;
(2) weighing disodium adenosine diphosphate according to the formula amount, pouring the disodium adenosine diphosphate into a freeze-drying protective agent solution, and uniformly mixing to obtain adenosine diphosphate detection reagent mother liquor;
(3) subpackaging the adenosine diphosphate detection reagent into penicillin bottles according to 40 microliter per bottle to obtain a finished product adenosine diphosphate detection reagent;
(4) and (3) carrying out vacuum freeze drying on the adenosine diphosphate detection reagent to obtain an adenosine diphosphate reagent freeze-dried product.
Example 2: the total amount of the adenosine diphosphate cup detection reagent in the embodiment is 60 microliters, and the dosage of each raw material according to the volume ratio is respectively: disodium adenosine diphosphate 0.02%, bovine serum albumin 3.0%, trehalose 2.0%, and the balance of distilled water.
The preparation method comprises the following steps:
(1) weighing bovine serum albumin and trehalose according to the formula amount, and adding distilled water or purified water to fully dissolve to obtain a freeze-drying protective agent solution;
(2) weighing disodium adenosine diphosphate according to the formula amount, pouring the disodium adenosine diphosphate into a freeze-drying protective agent solution, and uniformly mixing to obtain adenosine diphosphate detection reagent mother liquor;
(3) subpackaging 60 microliters of adenosine diphosphate detection reagent into penicillin bottles to obtain finished adenosine diphosphate detection reagent;
(4) and (3) carrying out vacuum freeze drying on the adenosine diphosphate detection reagent to obtain an adenosine diphosphate reagent freeze-dried product.
Example 3: the total amount of the adenosine diphosphate cup detection reagent in the embodiment is 40 microliters, and the dosage of each raw material according to the volume ratio is respectively as follows: disodium adenosine diphosphate 0.008%, bovine serum albumin 1.0%, trehalose 1.0%, and the balance of distilled water.
The preparation method comprises the following steps:
(1) weighing bovine serum albumin and trehalose according to the formula amount, and adding distilled water or purified water to fully dissolve to obtain a freeze-drying protective agent solution;
(2) weighing disodium adenosine diphosphate according to the formula amount, pouring the disodium adenosine diphosphate into a freeze-drying protective agent solution, and uniformly mixing to obtain adenosine diphosphate detection reagent mother liquor;
(3) subpackaging the adenosine diphosphate detection reagent into penicillin bottles according to 40 microliter per bottle to obtain a finished product adenosine diphosphate detection reagent;
(4) and (3) carrying out vacuum freeze drying on the adenosine diphosphate detection reagent to obtain an adenosine diphosphate reagent freeze-dried product.
Test example: the test example provides a reagent for detecting platelet aggregation functional adenosine diphosphate cup, the addition amount of disodium adenosine diphosphate is 0.001-0.02%, the dosage of the reagent is selected from ten levels of 0.002%, 0.004%, 0.006%, 0.008%, 0.01%, 0.012%, 0.014%, 0.016%, 0.018% and 0.02%, and the test groups are shown in table 1:
as can be seen from the data in table 2, the relative deviation of the measurement results of R, K, Angle and MA with the reference reagent in the test group No. 6 is the smallest, and the relative deviation is not more than 10%. Therefore, the amount of disodium adenosine diphosphate in the reagent of the present invention is preferably 0.012%.
The sugar freeze-drying protective agent added into the adenosine diphosphate cup detection reagent is one or more of trehalose, sucrose, glucan and glucose, and an experimental result shows that the freeze-drying effect is best when the trehalose accounting for 0.5-2.5% of the mass volume of the reagent is added, and the sugar freeze-drying protective agent has no obvious influence on all indexes of the adenosine diphosphate cup detection reagent. The trehalose without reducing group is added, so that the low-temperature dehydration protection effect can be realized, and the structure of the adenosine diphosphate disodium can be better maintained.
The polymer freeze-drying protective agent added into the adenosine diphosphate cup detection reagent is one or more of bovine serum albumin, polyvinylpyrrolidone and polyethylene glycol, and an experimental result shows that the freeze-drying effect is best when the bovine serum albumin with the mass volume of 0.1-3.0% of the reagent is added, and the polymer freeze-drying protective agent has no obvious influence on each index of the adenosine diphosphate cup detection reagent. Bovine serum albumin and trehalose are added to interact, so that the disodium adenosine diphosphate is prevented from deterioration and inactivation caused by low temperature and dehydration in the freeze-drying process.
Bovine serum albumin and trehalose are used as freeze-drying protective agents, and the effect of maintaining the activity of the disodium adenosine diphosphate is most ideal. After preliminary experiments, the dosage of each freeze-drying protective agent is temporarily determined as follows: trehalose was selected at three levels of 1.0%, 1.5% and 2.0%; bovine serum albumin was selected at three levels, 1.0%, 2.0% and 3.0%;
the test levels, factor combinations are shown in table 3 below.
Taking an imported reagent as a reference reagent, each sample is respectively carried out in parallel three times by using the reagent and the reference reagent, and the detection results are shown in the following table 4.
As can be seen from the data in table 4, the relative deviation of the measurement results of R, K, Angle and MA with the reference reagent in the No. 2 experimental group was the smallest, and the relative deviation was satisfied to be not more than 10%. Therefore, the amount of trehalose in the reagent of the present invention is preferably 1.0%, and the amount of bovine serum albumin in the reagent of the present invention is preferably 2.0%.
It can be seen that the most preferred embodiment of the present invention is: disodium adenosine diphosphate 0.012%, bovine serum albumin 2.0%, trehalose 1.0%, the rest is purified water.
The prepared adenosine diphosphate detection reagent can be used for whole blood detection and anticoagulation whole blood detection in the thromboelastogram technology.
Application example:
in the application example, the adenosine diphosphate detection reagent is used for the anticoagulation detection of the thromboelastogram, and the detection is as follows.
The Thromboelastogram (TEG) test feature quantities are explained below:
blood coagulation time R: the blood coagulation factors such as thrombin are sufficiently activated, fibrin clot formation starts, and the time is required until the amplitude reaches 2mm as measured by a thromboelastography. An extended R-value, indicative of a deficiency of certain coagulation factors; the decrease in R value indicates a high blood coagulation status.
Clot formation time K: the time from the end of the R value time to the time at which the thromboelastogram traces the amplitude to 20mm reflects the interaction of fibrin and platelets at the beginning of clotting formation. The length of the K value reflects the fibrinogen level.
Hemagglutination rate Angle: the thromboelastography instrument traces the included angle between the tangent line of the maximum radian point of the amplitude curve and the horizontal line, and reflects the forming speed of the blood clot. When the blood is in a severe low-coagulation state, the amplitude of the tracing meter does not reach 20mm, and the K value cannot be determined.
Blood clot strength MA: the maximum profilometer amplitude for the thromboelastogram, which is the binding of fibrin to the platelet GPIIb/IIIa receptor, represents the maximum strength of the fibrin/platelet clot.
Inhibition ratio%: less than 50, indicating that the medicine can not play a better anticoagulation effect and has a certain degree of medicine resistance.
Inhibition ratio%: more than or equal to 50, prompting the drug to take effect and obviously inhibit.
The adenosine diphosphate cup detection reagent provided by the invention is used for directly detecting anticoagulated whole blood treated by heparin sodium, and the collected anticoagulated whole blood is placed at room temperature for not more than 24 hours, preferably within 2 hours.
Disodium adenosine diphosphate: SIGMA;
trehalose manufacturers: shanghai-sourced leaf Biotechnology, Inc.;
bovine serum albumin manufacturers: BioRuler;
weighing bovine serum albumin and trehalose with specified amount of the formula, adding into a volumetric flask, adding purified water, and fully dissolving to obtain the freeze-drying protective agent solution.
And weighing adenosine diphosphate disodium with a specified amount in the formula, adding the disodium adenosine diphosphate into the freeze-drying protective agent solution, and fixing the volume by using purified water.
And (3) freeze-drying the prepared adenosine diphosphate cup detection reagent, and storing at 2-8 ℃ after the freeze-drying is finished.
The detection method is as follows.
1) Opening application software, inputting a patient name on a patient information interface, and selecting a test type, wherein the test type is a common cup ' CK-Cirtated kaolin ', a-Activated ' batroxobin cup ' and ' ADP-Activated + ADP ' adenosine diphosphate cup ';
2) loading a common cup, a batroxobin cup and an adenosine diphosphate cup on a channel of the thrombelastogram instrument; note and test type corresponds!
3) Taking out the batroxobin cup detection reagent, the adenosine diphosphate cup detection reagent and the purified water, respectively transferring 40 mu l of the purified water, adding the reagents of the batroxobin cup detection reagent and the adenosine diphosphate cup detection reagent into bottles, covering the bottles, and fully shaking up;
4) transferring 1ml of the anticoagulated whole blood sample of sodium citrate into a reagent 1 bottle by using an lml pipettor, turning the reagent 1 bottle upside down for 5 times, fully mixing uniformly, and standing for 4 minutes to activate blood;
5) and (3) detecting a common cup: transferring 20 mul of reagent 2 to the bottom of a common cup, transferring 340 mul of blood activated in the step 4) to the common cup, pinching a cup holder, pushing the common cup to the top end, shifting a test rod to a test position, selecting a CK-circled kaolin channel, clicking a software interface start or a keyboard F10, and starting testing;
6) batroxobin cup detection: transferring 10 mu l of liquid from the redissolving reagent A in the step 2) into a batroxobin cup, adding 360 mu l of heparinized blood sample, pumping three times in the cup, uniformly mixing, pushing the batroxobin cup to the top end, shifting a test rod to a test position, selecting an A-Activated channel, clicking a software interface 'start' or a keyboard F10, and starting testing;
7) detecting adenosine diphosphate in a cup: transferring 10 mul of liquid from the redissolving reagent A in the step 2), sucking 10 mul of liquid from the redissolving reagent C, adding the liquid into an adenosine diphosphate cup, adding 360 mul of heparinized blood sample, sucking for three times in the cup, uniformly mixing, pushing the adenosine diphosphate cup to the top end, shifting a test rod to a test position, selecting an ADP-Activated + ADP channel, clicking a software interface 'start' or a keyboard F10, and starting to test;
8) the clotting process was finished for about 30 minutes;
9) after the test is finished, clicking stop on a software interface, unloading the test cup, and disposing according to the laboratory management regulation;
10) inhibition rate: and clicking 'compounding' on a software interface, selecting a test result graph of three channels of a common cup, a batroxobin cup and an adenosine diphosphate cup, and clicking 'finishing' to check the inhibition rate information of the adenosine diphosphate pathway.
11) And after the detection is finished, closing the instrument power switch.
The results are shown in tables 5 to 9.
As can be seen from tables 6 and 7, the results of the anticoagulated whole blood tests of 3 random healthy people are all within the specified range, which indicates that the test results of the kit of the invention are consistent with the actual conditions of patients.
The results of the measurement of the in-batch precision and the inter-batch precision of the adenosine diphosphate detection reagent of the present invention are shown in tables 8 and 9.
As can be seen from Table 8, the variation coefficients of the precision detection results in batch for the fresh sodium citrate and heparin sodium anticoagulated whole blood of the common cup, the batroxobin cup and the adenosine diphosphate cup are all less than 10%, and the precision in batch is high.
As can be seen from Table 9, the relative range of the measurement results of the inter-lot precision of each characteristic amount is less than 10%, indicating that the inter-lot precision is high.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and scope of the present invention are intended to be covered thereby.
Claims (9)
1. A platelet aggregation function adenosine diphosphate cup detection reagent is characterized in that: comprises disodium adenosine diphosphate and a freeze-drying protective agent.
2. The reagent for detecting platelet aggregation functional adenosine diphosphate according to claim 1, wherein: the addition amount of the adenosine diphosphate is 0.012%.
3. The reagent for detecting platelet aggregation functional adenosine diphosphate according to claim 1, wherein: the freeze-drying protective agent comprises a saccharide freeze-drying protective agent and a polymer freeze-drying protective agent.
4. The reagent for detecting platelet aggregation functional adenosine diphosphate cup according to claim 3, wherein: the saccharide freeze-drying protective agent is one or more of trehalose, sucrose, glucan and glucose.
5. The reagent for detecting platelet aggregation functional adenosine diphosphate according to claim 4, wherein: the saccharide protective agent is trehalose, and the addition amount of trehalose is 1%.
6. The reagent for detecting platelet aggregation functional adenosine diphosphate cup according to claim 3, wherein: the polymer freeze-drying protective agent is one or more of bovine serum albumin, polyvinylpyrrolidone and polyethylene glycol.
7. The reagent for detecting platelet aggregation functional adenosine diphosphate according to claim 6, wherein: the polymer freeze-drying protective agent is bovine serum albumin, and the addition amount of the bovine serum albumin is 2%.
8. A method for preparing a reagent for assaying platelet aggregation-functional adenosine diphosphate according to any one of claims 1 to 7, which comprises: the method comprises the following steps:
(1) weighing the freeze-drying protective agent according to the formula amount, and adding distilled water or purified water to fully dissolve to obtain a freeze-drying protective agent solution;
(2) weighing disodium adenosine diphosphate according to the formula amount, pouring the disodium adenosine diphosphate into a freeze-drying protective agent solution, and uniformly mixing to obtain adenosine diphosphate detection reagent mother liquor;
(3) subpackaging the adenosine diphosphate detection reagent into penicillin bottles according to 20-60 microliter per bottle to obtain a finished product adenosine diphosphate detection reagent;
(4) and (3) carrying out vacuum freeze drying on the adenosine diphosphate detection reagent to obtain an adenosine diphosphate reagent freeze-dried product.
9. The method for preparing a reagent for detecting platelet aggregation functional adenosine diphosphate according to claim 8, wherein: the adenosine diphosphate detection reagent is subpackaged into penicillin bottles according to 40 microliter per bottle.
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