A heparin detection kit and its application

Abstract

The present invention discloses a heparin detection kit and its application, relating to the technical field of anticoagulation effect detection. Specifically, the heparin detection kit includes a heparin detection reagent, and the heparin detection reagent includes heparinase I. Heparinase I is a liquid enzyme preparation, and it needs to be freeze-dried and stored as a solid for long-term preservation. Adding a certain amount of anhydrous calcium chloride, glycerol, and D-trehalose to heparinase I can be used as a protective agent to make the detection reagent exist stably for a long time, meeting the requirement of long-term preservation. The heparin detection kit can quickly and specifically neutralize the anticoagulant activity of heparin without affecting other blood components involved in blood coagulation, so as to more accurately and quickly evaluate the residual situation of sodium heparin and the anticoagulation effect of sodium heparin, providing a basis for clinical drug use.

Description

Technical Field

[0001] The present invention relates to the technical field of anticoagulation effect detection, and in particular, to a heparin detection kit and its application. Background Art

[0002] Sodium heparin is an anticoagulant and is the preferred drug for rapidly achieving anticoagulation. Currently, it can be used clinically for preventing thrombosis in surgery and anticoagulant treatment of pregnant women.

[0003] For patients with acute myocardial infarction, heparin can be used to prevent venous embolism in patients and to prevent arterial embolism in patients with large anterior wall transmural myocardial infarction, and is used to maintain the patency of extracorporeal blood circulation during heart surgery and kidney dialysis. It is used to treat disseminated intravascular coagulation (DIC) caused by various reasons, and is also used to treat glomerulonephritis, nephrotic syndrome, rheumatoid arthritis, etc.

[0004] Currently, the evaluation of heparin anticoagulation effect in clinical practice is mainly carried out by APTT (activated partial thromboplastin time). The presence of sodium heparin in the blood will cause an extension of APTT, but it is susceptible to congenital reduction of coagulation factors II, V, VIII, IX, XI, XII, fibrinogen deficiency, enhanced fibrinolysis activity or other anticoagulants, which will affect the accuracy of the test results. Summary of the Invention

[0005] The first object of the present invention is to provide a heparin detection kit, which can quickly and specifically neutralize the anticoagulant activity of heparin without affecting other blood components involved in coagulation, so as to more accurately and quickly evaluate the residual amount of sodium heparin and the anticoagulation effect of sodium heparin, providing a basis for clinical medication.

[0006] The second object of the present invention is to provide a preparation method of a heparin detection reagent, which can effectively and quickly prepare a heparin detection reagent capable of accurately detecting the anticoagulation effect of heparin.

[0007] The third object of the present invention is to provide an activated coagulation detection kit, which includes a heparin detection reagent that can effectively and quickly prepare a heparin detection reagent capable of accurately detecting the anticoagulation effect of heparin.

[0008] The fourth object of the present invention is to provide the application of the above heparin detection reagent in thromboelastogram detection or coagulation detection, which can quickly and accurately detect the anticoagulation effect of heparin.

[0009] The present invention is implemented as follows:

[0010] An embodiment of the present invention provides a heparin detection kit, which includes a heparin detection reagent. The heparin detection reagent includes heparinase I, anhydrous calcium chloride, glycerol, and D-trehalose, wherein heparinase I is in a liquid state.

[0011] Heparinase I is a liquid enzyme preparation. For long-term preservation, it needs to be freeze-dried and stored in solid form. However, through long-term creative labor, the inventors of this patent combined anhydrous calcium chloride, glycerol, and D-trehalose with heparinase I, enabling heparinase I to be stably preserved in liquid form for a long time. This simplifies the operation process, speeds up the experimental progress, improves the detection efficiency, and at the same time reduces the transportation and storage costs, with strong practicability.

[0012] Specifically, in the heparin detection reagent, the concentration of heparinase I is 60%-80% (5 IU / ml - 10 IU / ml), the concentration of anhydrous calcium chloride is 0.05%-0.4%, the concentration of glycerol is 0.2%-0.8%, and the concentration of D-trehalose is 0.2%-0.8%.

[0013] The embodiment of the present invention also provides a preparation method of a heparin detection reagent, which includes the following steps:

[0014] Mix heparinase I, anhydrous calcium chloride, glycerol, and D-trehalose.

[0015] Specifically, the concentration of heparinase I is 60%-80% (5 IU / ml - 10 IU / ml), the concentration of anhydrous calcium chloride is 0.05%-0.4%, the concentration of glycerol is 0.2%-0.8%, and the concentration of D-trehalose is 0.2%-0.8%.

[0016] Furthermore, the embodiment of the present invention also provides an activated coagulation detection kit, which includes a heparin detection reagent and an activated coagulation detection reagent.

[0017] Specifically, the heparin detection reagent includes heparinase I, anhydrous calcium chloride, glycerol, and D-trehalose, wherein heparinase I is in liquid state.

[0018] The ratio and concentration of the components in this heparin detection reagent are the same as above and will not be elaborated here.

[0019] The embodiment of the present invention also provides the application of the above heparin detection kit or the above activated coagulation detection kit in thromboelastogram detection or coagulation detection.

[0020] Specifically, thromboelastogram (TEG) detection is an analyzer that monitors the coagulation process from the entire dynamic processes such as platelet aggregation, coagulation, and fibrinolysis, and is used to monitor and analyze the coagulation state of blood samples.

[0021] Its principle is based on the fact that the final result of the coagulation process is the formation of a blood clot, and the physical properties of the blood clot (blood clot strength and stability) determine whether it has normal coagulation function.

[0022] The R value is a detection parameter of a thromboelastograph. R is the time for the formation of initial fibrin. An extended R value indicates the use of anticoagulants or a deficiency of coagulation factors. A shortened R value indicates a hypercoagulable state of the blood.

[0023] In the embodiments of the present invention, the thromboelastograph detection includes a first-channel detection and a second-channel detection:

[0024] The first-channel detection is to detect the sample to be tested after being induced by an activated coagulation detection reagent. This detection is a normal cup detection, and the measured R 普通杯 ;

[0025] The second-channel detection is to detect the sample to be tested after being induced by the activated coagulation detection reagent after mixing it with the heparin detection reagent in the above heparin detection kit or the heparin detection reagent in the above activated coagulation detection kit. This detection is a heparinase cup detection, and the measured R 肝素酶杯 ;

[0026] Then, based on the measured R 普通杯 and R 肝素酶杯 judge the influence of heparin drugs on blood coagulation.

[0027] Specifically, during the process of judging the influence of heparin drugs on blood coagulation:

[0028] When |R 普通杯 −R 肝素酶杯 | ≤ 1 min, it is judged that the blood sample to be tested is not affected by heparin substances;

[0029] When R 肝素酶杯 < R 普通杯 , and R 普通杯 −R 肝素酶杯 ≥ 1 min, it is judged that the sample to be tested is affected by heparin substances.

[0030] Furthermore, the above coagulation detection is an activated partial thromboplastin time detection.

[0031] The present invention has the following beneficial effects:

[0032] The embodiments of the present invention provide a heparin detection kit and a preparation method thereof. The heparin detection kit includes a heparin detection reagent. The heparin detection reagent includes heparinase I. Heparinase I is a liquid enzyme preparation and needs to be freeze-dried and stored as a solid for long-term preservation. Adding a certain amount of anhydrous calcium chloride, glycerol, and D-trehalose to heparinase I can be used as a protective agent to make the detection reagent exist stably for a long time, meeting the requirement of long-term preservation.

[0033] This heparin detection kit can quickly and specifically neutralize the anticoagulant activity of heparin without affecting other blood components involved in blood coagulation, so as to more accurately and rapidly evaluate the residual amount of sodium heparin and the anticoagulant effect of sodium heparin, providing a basis for clinical medication.

[0034] An embodiment of the present invention also provides an activated coagulation detection kit and its application. The activated coagulation detection kit includes components that can quickly and specifically neutralize the anticoagulant activity of heparin without affecting other blood components involved in blood coagulation. Applying this activated coagulation detection kit to thromboelastography or coagulation detection can more accurately and effectively detect the residual amount of sodium heparin to evaluate the anticoagulant effect of sodium heparin. Detailed implementation manners

[0035] To make the objectives, 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. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0036] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0037] Embodiment 1

[0038] This embodiment provides a preparation method for a heparin detection reagent, and the preparation method includes the following steps:

[0039] The heparin detection reagent includes: heparinase I, anhydrous calcium chloride, glycerol, and D-trehalose; among them, heparinase I is in liquid form.

[0040] Specifically, the concentration of heparinase I is 8 IU / mL, the concentration of anhydrous calcium chloride is 0.2%, the concentration of glycerol is 0.6%, and the concentration of D-trehalose is 0.6%.

[0041] Embodiment 2

[0042] This embodiment provides a preparation method for a heparin detection reagent. The preparation method is substantially the same as the method provided in Embodiment 1, except for the differences in components, which are as follows:

[0043] The heparin detection reagent includes, by mass percentage: heparinase I, anhydrous calcium chloride, glycerol, and D-trehalose; among them, heparinase I is in liquid form.

[0044] Specifically, the concentration of heparinase I is 7 IU / mL, the concentration of anhydrous calcium chloride is 0.2%, the concentration of glycerol is 0.5%, and the concentration of D-trehalose is 0.6%.

[0045] Example 3

[0046] This example provides a preparation method for a heparin detection reagent. This preparation method is generally the same as the methods provided in Examples 1 and 2, except for the components, which are as follows:

[0047] The heparin detection reagent, by mass percentage, includes: heparinase I, anhydrous calcium chloride, glycerol, and D-trehalose; among them, heparinase I is in liquid form.

[0048] Specifically, the concentration of heparinase I is 7 IU / mL, the concentration of anhydrous calcium chloride is 0.3%, the concentration of glycerol is 0.4%, and the concentration of D-trehalose is 0.4%.

[0049] Example 4

[0050] This example provides the application of a heparin detection reagent in thromboelastogram detection.

[0051] In this example, the heparin detection reagent is the heparin detection reagent prepared by the preparation method provided in Example 1.

[0052] (1) Load sample cups on both the first channel and the second channel of the thromboelastogram instrument, and add 20 μL of 0.2 mol / L calcium chloride solution to the sample cups in both channels;

[0053] (2) Take 1 mL of uncentrifuged citrated anticoagulated whole blood (test blood sample), mix the test blood sample with an activated coagulation detection reagent (which is a prior art and will not be elaborated here), invert it 5 times up and down, and then let it stand for 1 min to obtain a test sample;

[0054] (3) Aspirate 340 μL of the test sample obtained in step (2) into the sample cup of the first channel in step (1), and click the "Start" button in the thromboelastogram instrument software according to the conventional operation steps to start the detection. The measured R value is R 普通杯 ;

[0055] (4) Aspirate 500 μL of the test sample obtained in step (2), add it to a reagent tube containing the heparin detection reagent, invert it 3 times up and down, let it stand for 1 min, then aspirate 340 μL of the test sample in the reagent tube into the sample cup of the second channel in step (1), and click the "Start" button in the thromboelastogram instrument software according to the conventional operation steps to start the detection. The measured R value is R 肝素酶杯 ;

[0056] (5) When the detections of both the first channel and the second channel are completed or the required parameter values are obtained, stop the detection according to the conventional operation process.

[0057] (6) Result judgment

[0058] When R 肝素酶杯﹤R 普通杯 and R 普通杯 -R 肝素酶杯 ≥1 min, it is determined that the sample to be tested is affected by heparin-like substances, and the coagulation ratio is lower than that before being affected by heparin-like substances;

[0059] When |R 普通杯 -R 肝素酶杯 |≤1 min, it is determined that the tested blood sample is not affected by heparin-like substances, and the coagulation is close to that before being affected by heparin-like substances.

[0060] Comparative Example 1

[0061] The performance of the heparin detection reagent provided in Example 1.

[0062] Verify the heparin detection reagent prepared by the preparation method provided in Example 1, whose storage time can be up to 18 months. Compare the heparin detection reagent provided in this Example 1 with the heparin detection reagent provided by the prior art (Lepu heparin detection reagent).

[0063] Experimental method

[0064] Put 3 - 6 IU of sodium heparin into 1 mL of blood and mix evenly. Take 500 μL of heparinized blood sample and place it in the reagent and mix well to compare the neutralization ability.

[0065] Generally, after adding sodium heparin, the blood does not coagulate and the R value cannot be determined. However, after using the reagent provided by the present invention to neutralize sodium heparin, the blood sample restores normal coagulation, and the measured R value is 4 - 11.

[0066] The embodiment of the present invention provides a heparin detection kit, which includes a heparin detection reagent. The heparin detection reagent includes heparinase I, and heparinase I is a liquid enzyme preparation. For long-term storage, it needs to be freeze-dried and stored as a solid. And adding a certain amount of anhydrous calcium chloride, glycerol and D-trehalose to heparinase I can be used as a protective agent to make the detection reagent exist stably for a long time, meeting the requirement of long-term storage.

[0067] This heparin detection kit can quickly and specifically neutralize the anticoagulant activity of heparin without affecting other blood components of coagulation, so as to more accurately and quickly evaluate the residual situation of sodium heparin and the anticoagulant effect of sodium heparin, providing a basis for clinical medication.

[0068] The embodiment of the present invention also provides an activated coagulation detection kit and its application. This activated coagulation detection kit can quickly and specifically neutralize the anticoagulant activity of heparin without affecting other blood components of coagulation. And applying this activated coagulation detection kit to thromboelastography or coagulation detection can more accurately and effectively detect the residual situation of sodium heparin to evaluate the anticoagulant effect of sodium heparin.

[0069] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A heparin detection kit, characterized in that, It includes a heparin detection reagent, and the heparin detection reagent is composed of heparinase I, anhydrous calcium chloride, glycerol and D-trehalose; wherein, the heparinase I is in liquid state; The concentration of the heparinase I is 5 IU / ml - 10 IU / ml, the concentration of the anhydrous calcium chloride is 0.05% - 0.4%, the concentration of the D-trehalose is 0.2% - 0.4%, and the concentration of the glycerol is 0.2% - 0.4%.

2. The application of the heparin detection kit according to claim 1 in thromboelastogram detection or coagulation detection.

3. The application according to claim 2, characterized in that, The coagulation detection includes activated partial thromboplastin time detection.

4. An activated clotting detection kit, characterized in that, It includes a heparin detection reagent; the heparin detection reagent is composed of heparinase I, anhydrous calcium chloride, glycerol and D-trehalose; the heparinase I is in liquid state; The concentration of the heparinase I is 5 IU / ml - 10 IU / ml, the concentration of the anhydrous calcium chloride is 0.05% - 0.4%, the concentration of the D-trehalose is 0.2% - 0.4%, and the concentration of the glycerol is 0.2% - 0.4%.

5. The activated coagulation detection kit according to claim 4, wherein The activated coagulation detection kit includes an activated coagulation detection reagent.

6. The application of the activated coagulation detection kit according to claim 4 in thromboelastogram detection or coagulation detection.

7. The application according to claim 6, wherein The coagulation detection includes activated partial thromboplastin time detection.