Two-level coagulation activation test reagent quality control product and preparation method and application thereof

Abstract

The present application relates to the technical field of coagulation test quality control, in particular to two-level coagulation activation test reagent quality control products and preparation method and application thereof. The two-level quality control products include coagulation activation quality control product level 1 with TEG- ACT value of 100±20S and MA value of 60±15mm, and coagulation activation quality control product level 2 with TEG- ACT value of 160±30S. The two-level quality control products cover normal coagulation state and abnormal coagulation state, and are used in combination with thrombelastograph and coagulation activation reagent, so as to control the quality of thrombelastogram coagulation activation detection system or evaluate the detection system. The coagulation activation quality control product level 2 is particularly sensitive to abnormal conditions of matched coagulation activation reagent, and can be used for monitoring abnormal conditions of matched reagent. The present application solves the technical problem that the prior art lacks stable and reliable quality control products for coagulation activation test reagents, is easy to industrialize, and has an ideal application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of coagulation test quality control, in particular to two-level coagulation activation test reagent quality control products and a preparation method and application thereof. BACKGROUND

[0002] Thromboelastography is a curve of coagulation dynamic process drawn by a thromboelastograph, and is a curve graph capable of dynamically analyzing the whole process of coagulation formation and fibrinolysis. The thromboelastograph is usually used together with a matching reagent to detect a clinical sample, so as to achieve different detection purposes.

[0003] Ordinary activation of thromboelastography uses thromboelastography test (activated coagulation) reagent (coagulation method), only triggers the endogenous coagulation pathway, and the coagulation time of a normal sample is 5-10 minutes according to the thromboelastography detection result. Thromboelastography coagulation activation, also known as rapid activation of thromboelastography (r-TEG), uses thromboelastography test (coagulation activation) reagent (coagulation method), can simultaneously activate the endogenous and exogenous coagulation pathways, and the coagulation time is shorter (normal value ≤ 2 minutes), thus being more beneficial to the diagnosis and real-time monitoring of coagulation dysfunction after trauma in an emergency department. The coagulation activation detection system (rapid activation detection system) is composed of a detection device (thromboelastograph) and coagulation activation test reagent (rapid activation test reagent), and is used for detecting a clinical sample. The detection process is roughly as follows: first, the coagulation activation test reagent is added to the clinical sample, and then the thromboelastograph is used for detection, so as to obtain related coagulation parameters. Before the detection system is used for detecting a clinical sample, a suitable quality control product is used to evaluate whether the detection system is in a normal state, so as to ensure the reliability of the detection result of the clinical sample. At present, there is no stable and reliable coagulation activation test reagent quality control product on the market, and therefore it is necessary to develop a quality control product suitable for the detection system. SUMMARY

[0004] The present application aims to provide two-level coagulation activation test reagent quality control products, so as to solve the technical problem that there is no stable and reliable quality control product for coagulation activation test reagent in the prior art.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The two-level coagulation activation test reagent quality control product comprises coagulation activation quality control product level 1 with a TEG-ACT value of 100±20S and a MA value of 60±15mm, and coagulation activation quality control product level 2 with a TEG-ACT value of 160±30S.

[0007] The present application also provides a preparation method of the two-level coagulation activation test reagent quality control product, comprising the following steps performed in sequence:

[0008] S1: mixing the plasma matrix solution, fibrinogen matrix solution, anticoagulant, pH buffer, protein protective agent and preservative to obtain a stock solution;

[0009] S2: adjusting the TEG-ACT value and / or MA value of the stock solution to a specified range to obtain a freeze-drying stock solution, and then performing freeze-drying treatment to obtain the quality control product.

[0010] The present application also provides an application of the two-level coagulation activation detection reagent quality control product in evaluating the stability and accuracy of a coagulation activation detection system, wherein the coagulation activation detection system comprises a thromboelastograph and a coagulation activation detection reagent.

[0011] The principle and advantages of the present application are as follows:

[0012] The quality control product is usually used before actual sample detection to determine the stability of the detection system, thereby ensuring the accuracy of the actual sample detection result. At present, there is no quality control product suitable for the coagulation activation detection system on the market. The quality control product of the present application can realize quality control of the coagulation activation detection system comprising a thromboelastograph and a coagulation activation detection reagent. In the present application, the product with a TEG-ACT value of 100±20S and an MA value of 60±15mm can be used as coagulation activation quality control product level 1. The coagulation activation quality control product level 1 can simulate a normal coagulation state and perform quality control on the coagulation activation detection system comprising a thromboelastograph and a matching reagent. In the present application, the product with a TEG-ACT value of 160±30S can be used as coagulation activation quality control product level 2. The coagulation activation quality control product level 2 can simulate an abnormal coagulation state and perform quality control on the coagulation activation detection system comprising a thromboelastograph and a matching reagent. The matching reagent can be a coagulation activation detection system thromboelastography test (coagulation activation) reagent (coagulation method) produced by the present application, which simultaneously activates the endogenous and exogenous coagulation pathways and is used for diagnosis and real-time monitoring of coagulation dysfunction after trauma in the emergency department.

[0013] In the present application, both the coagulation activation quality control product level 1 and the coagulation activation quality control product level 2, when subjected to the thromboelastograph and the matching reagent to form a coagulation activation detection system, can obtain relevant coagulation parameters, and the variation coefficients of the TEG-ACT value, Angle value and MA value after multiple measurements are small, meeting the application requirements. The quality control product can be used to evaluate whether the coagulation activation detection system is in an abnormal state. The quality control product is subjected to a strict assignment experiment to obtain its assignment range. If the detection result of the to-be-detected coagulation activation detection system is within the assignment range, it can be determined that the system is normal. Otherwise, it is not normal, and the thromboelastograph and the matching reagent need to be detected separately to exclude faults. In addition, the quality control product prepared in the present application also shows stable performance in the accelerated experiment, and is suitable for application and promotion.

[0014] In addition, the coagulation activator control level 2 is particularly sensitive to abnormal conditions of the matching reagent, and the TEG-ACT value of the coagulation activator control level 2 can change significantly with the content of the functional component in the matching reagent. By observing the changes, we can more accurately determine whether the matching reagent has failed. In actual application, we can compare the unknown matching reagent that may have failed with the qualified matching reagent that has just been factory inspected, and use the coagulation activator control level 2 as the control. If the detection result of the TEG-ACT value of the unknown matching reagent deviates greatly from the detection result of the TEG-ACT value of the qualified matching reagent (for example, the relative deviation of the mean value is greater than or equal to 5%), we believe that the unknown matching reagent is in an abnormal state and needs to be replaced. After a large number of tests, it is found that the coagulation activator control level 2 has a relatively ideal and significant sensitivity to the content of the functional component of the matching reagent compared with the coagulation activator control level 1, normal human samples and other comparative controls. Comparative Example 2 also proves that the coagulation activator control level 2 (TEG-ACT value controlled at 160±30S) can effectively evaluate the abnormal conditions of the coagulation activation detection reagent (such as low content and failure), and also ensure the stability of the sample.

[0015] Further, the raw materials of the coagulation activator control level 1 all include plasma matrix liquid, fibrinogen matrix liquid, anticoagulant, pH buffer, protein protective agent and preservative.

[0016] Further, the raw materials of the coagulation activator control level 2 all include plasma matrix liquid, anticoagulant, pH buffer, protein protective agent and preservative.

[0017] The plasma matrix liquid and / or the fibrinogen matrix liquid can constitute the coagulation main body. In order to ensure the stability of the product and the convenience of transportation, we usually need to freeze-dry the control. The addition of pH buffer, protein protective agent and preservative can ensure better product preservation effect. The protein protective agent plays a role in protecting the protein substances in the control; the pH buffer can provide a stable pH environment for various biomolecules in the control and maintain a certain salt ion concentration to maintain the active state of the biomolecules in the control; the use of preservatives can prevent the control from spoiling and prolong the shelf life of the control.

[0018] Further, the anticoagulant is at least one of bivalirudin, edoxaban and enoxaparin sodium.

[0019] When bivalirudin, edoxaban and enoxaparin sodium are used as anticoagulants, the uniformity and stability of the control are better, and the control can more accurately reflect whether the coagulation activation detection system is in an abnormal state.

[0020] The inventors found through a large number of studies that the use of different anticoagulants has a certain influence on the stability of the quality control product. By using different anticoagulants (sodium heparin, lithium heparin, sodium citrate, bivalirudin, edoxaban, enoxaparin sodium, etc.) to prepare the corresponding quality control product, the obtained quality control product is detected. The experimental results show that when sodium heparin or lithium heparin or sodium citrate or other anticoagulants are added, the stability of the product is poor, and the CV value (imprecision) of the detection result is large; while an appropriate amount of enoxaparin sodium or bivalirudin or edoxaban is added, the stability of the product is better, and the CV value (imprecision) of the detection result is smaller. Therefore, enoxaparin sodium or bivalirudin or edoxaban should be selected as the anticoagulant to prepare the coagulation activation quality control product.

[0021] Further, the protein protective agent includes at least one of bovine serum albumin, sucrose, trehalose, glycine, mannitol and lactose; the pH buffer is at least one of Hepes buffer, Tris-HCl buffer and phosphate buffer; and the preservative is at least one of Proclin 300, sodium azide and sodium benzoate. The above protein protective agent, pH buffer and preservative are conventional reagents in the prior art, which are stable in nature and moderate in cost.

[0022] Further, the plasma matrix liquid and the fibrinogen matrix liquid are prepared by the following method: sodium citrate is added to fresh pig whole blood to obtain anticoagulated pig blood; after the anticoagulated pig blood is allowed to stand and stratify, the upper liquid is taken, and then the supernatant is taken by centrifugation to obtain pig plasma; after the pig plasma is subjected to freeze-thaw and centrifugation, the upper layer is taken to obtain the plasma matrix liquid, and the lower layer is taken to obtain the fibrinogen matrix liquid.

[0023] By adopting the technical scheme, the fibrinogen matrix liquid and the plasma matrix liquid can be separated from pig blood, the above operation process is simple and easy to industrialize, and the scheme uses pig blood as the main raw material, which is abundant in source and low in cost.

[0024] Further, in S2, the TEG-ACT value of the stock solution is adjusted to 100±20S and the MA value is adjusted to 60±15mm to obtain coagulation activation quality control product level 1.

[0025] In the scheme, the product with the TEG-ACT value of 100±20S and the MA value of 60±15mm can be used as the coagulation activation quality control product level 1, the coagulation activation quality control product level 1 can simulate the normal coagulation state, and the coagulation activation detection system containing a thromboelastograph and matching reagents can be controlled.

[0026] Further, in S2, the TEG-ACT value of the stock solution is adjusted to 160±30S to obtain coagulation activation quality control product level 2.

[0027] In the scheme, the product with TEG-ACT value of 160±30S can be used as coagulation activation control level 2, which can simulate abnormal coagulation state and control the coagulation activation detection system containing a thrombelastograph and matching reagents.

[0028] In summary, the beneficial effects of the present scheme are:

[0029] At present, there is no suitable control product for the coagulation activation detection system on the market, so it is impossible to evaluate whether the detection system is in a normal state, and the reliability of the clinical sample detection result cannot be guaranteed. The present scheme overcomes the defects of the prior art and provides a coagulation activation detection reagent control product that can simulate the actual clinical detection environment and cover normal coagulation and abnormal coagulation. The coagulation activation control level 2 is better than the human sample, and can more effectively evaluate the abnormal condition of the coagulation activation detection reagent. The user can conveniently control the quality of the coagulation activation detection system or evaluate the detection system, and ensure the reliability of the clinical sample detection result. The present invention uses pig blood as the main raw material, which is abundant in source and low in cost; the preparation process is simple, the product is stable, and the industrial production is easy. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The detection spectrum of Table 1 Result 1 of Example 1 of the present invention.

[0031] Figure 2 The detection spectrum of Table 2 Result 1 of Example 1 of the present invention after 37℃ acceleration for 7 days.

[0032] Figure 3 The detection spectrum of Table 3 Result 1 of Example 2 of the present invention.

[0033] Figure 4 The detection spectrum of Table 4 Result 1 of Example 2 of the present invention after 37℃ acceleration for 7 days.

[0034] Figure 5 The detection spectrum of Table 5 Result 1 of the present invention for 20220614-1 batch.

[0035] Figure 6 The detection spectrum of Table 5 Result 1 of the present invention for 20220614-2 batch.

[0036] Figure 7 The detection spectrum of Table 5 Result 1 of the present invention for 20220614-3 batch.

[0037] Figure 8 The detection spectrum of Table 5 Result 1 of the present invention for 20220614-4 batch.

[0038] Figure 9The detection map of 8 μL of one result of the 20220614-5 batch in Table 5 of Invention Comparative Example 1.

[0039] Figure 10 The detection map of 8 μL of one result of the 20220614-6 batch in Table 5 of Invention Comparative Example 1.

[0040] Figure 11 The detection map of 8 μL of one result of the normal human source sample in Table 6 of Invention Comparative Example 2.

[0041] Figure 12 The detection map of 8 μL of one result of the 20220301 batch of coagulation activation quality control sample level 1 in Table 6 of Invention Comparative Example 2.

[0042] Figure 13 The detection map of 8 μL of one result of the 20220301 batch of coagulation activation quality control sample level 2 in Table 6 of Invention Comparative Example 2.

[0043] Figure 14 The detection map of 8 μL of one result of the 20220101 batch of comparative quality control sample in Table 6 of Invention Comparative Example 2.

[0044] Figure 15 The detection map of 8 μL of one result of the 20220614 batch of comparative quality control sample in Table 6 of Invention Comparative Example 2. DETAILED DESCRIPTION

[0045] The application will be further described in conjunction with the examples below, but the embodiments of the application are not limited thereto. If not specifically indicated, the technical means used in the following examples are conventional means known to those skilled in the art; the experimental methods used are conventional methods; and the materials, reagents, etc. used can be obtained from commercial channels.

[0046] In the examples and comparative examples of the present scheme, the main detection instruments, supporting reagents, and raw material information involved are as follows:

[0047] DRNX-III thromboelastograph: Chongqing Dingrun Medical Device Co., Ltd.; thromboelastometry test (coagulation activation) reagent (coagulation method): Chongqing Dingrun Medical Device Co., Ltd. (Registration No.: Yu Xie Zhuzhu 20192400263); calcium chloride solution: Chongqing Dingrun Medical Device Co., Ltd.; anticoagulated pig blood: Chongqing Shag Ecological Agriculture Development Co., Ltd.; sucrose: Sinopharm Chemical Reagent Co., Ltd.; glycine: Sinopharm Chemical Reagent Co., Ltd.; sodium chloride: Sinopharm Chemical Reagent Co., Ltd.; sodium citrate dihydrate: Sinopharm Chemical Reagent Co., Ltd.; Proclin 300: Sigma; sodium azide: Sigma; 1M Hepes: Beijing Solebao Technology Co., Ltd.; Heparin sodium: Hebei Changshan Biochemical Pharmaceutical Co., Ltd.; Heparin lithium: Hebei Changshan Biochemical Pharmaceutical Co., Ltd.; Bivalirudin: Chengdu Yunxi Chemical Co., Ltd.; Edoxaban: Shanghai Langxu Biotechnology Co., Ltd.; Enoxaparin sodium: Hebei Changshan Biochemical Pharmaceutical Co., Ltd.; Fibrinogen matrix fluid: Chongqing Dingrun Medical Equipment Co., Ltd.; Plasma matrix fluid: Chongqing Dingrun Medical Equipment Co., Ltd.

[0048] Example 1: Coagulation Activation Control Level 1 (Normal Value Control)

[0049] 1. Preparation of Plasma Matrix and Fibrinogen Matrix

[0050] (1) Collection of anticoagulated pig blood: When the pigs were slaughtered, fresh pig whole blood was mixed with 3.8% sodium citrate anticoagulant at a volume ratio of 9:1 to collect anticoagulated pig blood.

[0051] (2) Preparation of porcine plasma: Collect anticoagulated porcine blood and place it at room temperature for 3 to 6 hours to allow it to separate into layers. Aspirate the upper layer of liquid and centrifuge it in a tubular centrifuge. Set the centrifugation parameters as follows: speed 4500 rpm, time 15 min, temperature 4°C. After centrifugation, carefully aspirate the upper layer to obtain porcine plasma and freeze it at -20 to -30°C (valid for 3 years). The lower layer mainly contains red blood cells and should be treated as medical waste.

[0052] (3) Preparation of plasma matrix fluid and fibrinogen matrix fluid: Place the pig plasma that has been frozen into hard ice cubes in an environment of 2-8°C and slowly thaw until a few crushed ice cubes remain. Place the pig plasma in a tubular centrifuge and centrifuge with the following parameters: speed 4500 rpm, time 15 min, temperature 4°C. After centrifugation, carefully aspirate the upper layer (the plasma matrix fluid) and freeze it at -20--30°C (valid for 3 years); collect the lower layer (the fibrinogen matrix fluid) and freeze it at -20--30°C (valid for 3 years).

[0053] 2. Preparation of Coagulation Activation Control Level 1 (Normal Value Control)

[0054] (1) Take out the frozen fibrinogen matrix liquid and plasma matrix liquid, and place them in a 37±2℃ water bath for rapid thawing. In a 2000ml beaker, add 919.85ml of thawed plasma matrix liquid, 50ml of thawed fibrinogen matrix liquid, 0.05ml of 1% (mass fraction) enoxaparin sodium solution (anticoagulant), 10ml of 1M Hepes solution (pH buffer), 10ml of 1M sucrose solution (protein protective agent), 10ml of 1M glycine solution (protein protective agent), and 0.1ml of Proclin 300 (preservative) in sequence, stir for 15min, and obtain the coagulation activator control level 1 stock solution. The anticoagulant, pH buffer, protein protective agent, and preservative are collectively referred to as lyophilization protective agent, and the total amount is 30.15ml.

[0055] In actual operation, different anticoagulants, pH buffers, protein protective agents, and preservatives can be used: the protein protective agent includes at least one of bovine serum albumin, sucrose, trehalose, glycine, mannitol, and lactose; the pH buffer is at least one of Hepes buffer, Tris-HCl buffer, and phosphate buffer; and the preservative is at least one of Proclin 300, sodium azide, and sodium benzoate. The amount of anticoagulant added in the whole system is determined by the TEG-ACT value and the MA value, and the pH buffer, protein protective agent, and preservative are conventional lyophilization protective agents in the prior art, and their amounts can be determined by conventional experiments. The amounts of the above pH buffer, protein protective agent, and preservative are only used in this embodiment, and can be adjusted according to actual conditions in actual operation.

[0056] (2) Take the coagulation activator control level 1 stock solution, and use a thromboelastography instrument (such as DRNX-Ⅲ type) and a thromboelastography test (coagulation activation) reagent (coagulation method) to detect the TEG-ACT value and the MA value. The TEG-ACT value is 100±20S and the MA value is 60±15mm. Otherwise, supplement enoxaparin sodium solution to increase the TEG-ACT value and decrease the MA value, supplement fibrinogen matrix liquid to increase the MA value, and supplement plasma matrix liquid to decrease the TEG-ACT value and the MA value, until the TEG-ACT value and the MA value are adjusted to the above range, and the freeze-drying stock solution is obtained.

[0057] (3) The to-be-lyophilized stock solution is divided into 1 mL per bottle, and after 40 hours of lyophilization, the coagulation activator control product level 1 lyophilized product is obtained, which is stored at 2-8°C in a sealed state, and the effective period is 12 months. Each bottle of coagulation activator control product level 1 contains 1 mL of to-be-lyophilized stock solution, which is obtained by lyophilization. 1 mL of the stock solution contains approximately 0.92 mL of plasma matrix solution (919.85 mL / (50 mL+919.85 mL+30.15 mL)), 0.05 mL of fibrinogen matrix solution (50 mL / (50 mL+919.85 mL+30.15 mL)), and 0.03 mL of lyophilization protectant (30.15 mL / (50 mL+919.85 mL+30.15 mL)). When used, 1 mL of purified water is added to each bottle of lyophilized product to obtain a reconstituted solution.

[0058] 3. Performance detection of coagulation activator control product level 1

[0059] Detection method: According to the "Thromboelastogram Test (Coagulation Activation) Reagent (Coagulation Method) Instructions", add 10 μL of coagulation activation detection reagent (Thromboelastogram Test (Coagulation Activation) Reagent (Coagulation Method)) and 20 μL of 0.2M calcium chloride solution to the sample cup, then add 340 μL of control product reconstituted solution (add 1 mL of purified water to each bottle of control product to reconstitute), mix well by blowing and sucking 3 times, and start detection.

[0060] Appearance: The product is a white or light yellow solid, and after reconstitution, it is a colorless or light yellow liquid.

[0061] Uniformity: Use a thromboelastograph (such as DRNX-III) and a thromboelastogram test (coagulation activation) reagent (coagulation method) to detect according to the "Thromboelastogram Test (Coagulation Activation) Reagent (Coagulation Method) Instructions", and the detection results are shown in Table 1. The detection graph of "Result 1" in Table 1 is shown in Figure 1 . According to the detection results, the TEG-ACT value, Angle value and MA value of the product have small variation coefficients (CV), which meet the requirements (the variation coefficient (CV) should be ≤15%). In this experiment, in order to ensure that the experimental results can reflect the quality of the coagulation activator control product level 1, the thromboelastograph has been calibrated and qualified, and the thromboelastogram test (coagulation activation) reagent (coagulation method) has also been detected and qualified, and there is no failure or abnormality of the efficacy component content. This experiment is mainly to study whether there is a large deviation between the detection results of the control product and whether it has ideal uniformity when multiple samples in the same batch of control product are measured.

[0062] Table 1: Performance detection results of coagulation activator control product level 1 (20220301 batch)

[0063]

[0064]

[0065] 4. Stability test of coagulation activation control level 1

[0066] After the product was accelerated at 37℃ for 7 days (reference: China Pharmacopoeia 2015 Part III-P399: the product is estimated to have a shelf life of 1 year at 2-8℃), the thromboelastography instrument (such as DRNX-III) and thromboelastography test (coagulation activation) reagent (coagulation method) were used for detection, and the results are shown in Table 2. The detection map of "37℃ accelerated for 7 days after the results of 1" is shown in Figure 2 . The detection results show that the product still has detection potency (the relative deviation should be ≤15%) after being accelerated at 37℃ for 7 days. The relative deviation after being accelerated at 37℃ for 7 days = (the average value of detection after being accelerated at 37℃ for 7 days - the average value of detection before being accelerated at 37℃ for 7 days) / the average value of detection before being accelerated at 37℃ for 7 days x 100%.

[0067] Table 2: Stability test results of coagulation activation control level 1 (20220301 batch)

[0068]

[0069] Example 2: Coagulation activation control level 2 (abnormal value control)

[0070] 1. Preparation of coagulation activation control level 2 (abnormal value control)

[0071] (1) Take out the frozen plasma matrix solution and place it in a 37±2℃ water bath for rapid thawing. In a 2000mL beaker, add 969.4mL of thawed plasma matrix solution, 0.5mL of 1% enoxaparin sodium solution, 10mL of 1M Hepes solution, 10mL of 1M sucrose solution, 10mL of 1M glycine solution, and 0.1mL of Proclin300, stir for 15min, and obtain the coagulation activation control level 2 stock solution. The anticoagulant, pH buffer, protein protective agent and preservative are collectively referred to as lyophilization protective agent, which is 30.6ml in total.

[0072] (2) Take the coagulation activation control level 2 stock solution, and use the thromboelastography instrument (such as DRNX-III) and thromboelastography test (coagulation activation) reagent (coagulation method) for detection, and the TEG-ACT value is 160±30S, and the freeze-dried stock solution is obtained. Otherwise, adding enoxaparin sodium solution can increase TEG-ACT, and adding plasma matrix solution can reduce TEG-ACT.

[0073] (3) The to-be-lyophilized stock solution is divided into 1 mL per bottle, and after 40 hours of lyophilization, the coagulation activator control level 2 lyophilized product is obtained and stored at 2-8°C in a sealed state, with a valid period of 12 months. Each bottle of coagulation activator control level 2 contains 1 mL of to-be-lyophilized stock solution, which is obtained by lyophilization. 1 mL of the stock solution contains approximately 0.97 mL of plasma matrix solution (969.4 mL / (969.4 mL+30.6 mL)) and 0.03 mL of lyophilization protectant (30.6 mL / (969.4 mL+30.6 mL)). When used, 1 mL of purified water is added to each bottle of lyophilized product to obtain a reconstituted solution.

[0074] 2. Performance test of coagulation activator control level 2

[0075] Test method: The test method is the same as described in the performance test of coagulation activator control level 1.

[0076] Appearance: The product is a white or light yellow solid, and after reconstitution, it is a colorless or light yellow liquid.

[0077] Uniformity: The test results using a thromboelastography instrument (such as DRNX-III) and thromboelastography test (coagulation activation) reagent (coagulation method) are shown in Table 3. The test pattern of "result 1" in Table 3 is shown in Figure 3 From the test results, the MA value coefficient of variation (CV) of coagulation activator control level 2 is smaller, which meets the requirements (the coefficient of variation (CV) of MA value should be ≤15%).

[0078] Table 3: Performance test results of coagulation activator control level 2 (20220301 batch)

[0079] Detection parameter TEG-ACT(s) Angle (deg) MA (mm) Result 1 160.0 77.4 41.9 Result 2 152.0 78.5 42.5 Result 3 152.0 78.9 42.6 Result 4 152.0 76.5 41.4 Result 5 175.0 76.1 42.1 Result 6 160.0 77.2 42.4 Result 7 175.0 76.6 40.8 Result 8 175.0 75.5 42.0 Result 9 144.0 79.0 40.5 Result 10 167.0 77.2 42.0 CV 7.0％ 1.5％ 1.7％

[0080] 3. Stability test of coagulation activator control level 2

[0081] After the product is accelerated at 37°C for 7 days (reference: Chinese Pharmacopoeia 2015 Part III-P399: rough estimate of product at 2-8°C for 1 year), it is tested using a thromboelastography instrument (such as DRNX-III) and thromboelastography test (coagulation activation) reagent (coagulation method), and the results are shown in Table 4. The test pattern of "result 1 after 37°C acceleration for 7 days" is shown in Figure 4 The test results show that the product still has detection efficacy after 7 days of 37°C acceleration (the relative deviation should be ≤15%).

[0082] Table 4: Stability test results of coagulation activator control level 2 (20220301 batch)

[0083]

[0084] Comparative Example 1: Effect of different anticoagulants on the preparation of the control product

[0085] Referring to "Example 2: Coagulation activation quality control level 2 (outlier quality control)", different anticoagulants were added to the thawed plasma matrix solution, and the proportion of the anticoagulants was adjusted to a TEG-ACT value of about 200 s, and the other operations were the same as Example 2. The effect of different anticoagulants on the preparation of quality control was studied. The inventors found through a large number of studies that the addition of a certain amount of anticoagulant can adjust the TEG-ACT value to the required range. However, the use of different anticoagulants has a certain impact on the stability of the quality control. By using different anticoagulants (sodium heparin, lithium heparin, sodium citrate, bivalirudin, edoxaban, enoxaparin sodium, etc.) to prepare the corresponding quality control, the obtained quality control was detected, and the experimental results are shown in Table 5. The detection spectrum of "20220614-1 batch result 1 is shown in Figure 5 , the detection spectrum of 20220614-2 batch result 1 is shown in Figure 6 , the detection spectrum of 20220614-3 batch result 1 is shown in Figure 7 , the detection spectrum of 20220614-4 batch result 1 is shown in Figure 8 , the detection spectrum of 20220614-5 batch result 1 is shown in Figure 9 , the detection spectrum of 20220614-6 batch result 1 is shown in Figure 10 ".

[0086] The experimental results show that when heparin sodium or heparin lithium or sodium citrate or other anticoagulants are added, the stability of the product is poor, and the CV value (imprecision) of the detection result is large; while adding an appropriate amount of enoxaparin sodium or bivalirudin or edoxaban, the stability of the product is good, and the CV value (imprecision) of the detection result is small. Therefore, enoxaparin sodium or bivalirudin or edoxaban should be selected as the anticoagulant for preparing the coagulation activation quality control.

[0087] Table 5: TEG-ACT value detection results of samples using different anticoagulants

[0088] Batch number Result 1 Result 2 Result 3 Result 4 CV value Remark 20220614-1 355s 191s 245s 214s 28.9％ Add final concentration 15 pg / mL Heparin Sodium 20220614-2 206s 199s 277s 323s 23.6％ Add final concentration 15 pg / mL Heparin Lithium 20220614-3 191s 214s 323s 222s 24.6％ Add final concentration 25 pg / mL Sodium Citrate 20220614-4 206s 206s 191s 238s 9.4％ Add final concentration 2.5 pg / mL Bivalirudin 20220614-5 214s 230s 199s 191s 8.3％ Add final concentration 1.0 pg / mL Edoxaban 20220614-6 214s 222s 191s 206s 6.4％ Add final concentration 4.0 pg / mL Enoxaparin Sodium

[0089] Example 2: Coagulation activation quality control level 2 (outlier quality control)

[0090] Referring to "Example 2: Coagulation activation quality control level 2 (outlier quality control)", different proportions of enoxaparin sodium solution were added to the thawed plasma matrix solution, and other operations were the same as in Example 2, to obtain 20220101 batch comparative quality control with TEG-ACT value < 130S and 20220101 batch comparative quality control with TEG-ACT value > 200S, respectively. Using the same thrombelastograph (DRNX-III type thrombelastograph, which has been calibrated), according to "Thrombelastography test (coagulation activation) reagent (coagulation method) instructions", 8 μL, 10 μL, 12 μL coagulation activation detection reagent (thrombelastography test (coagulation activation) reagent (coagulation method)) and 20 μL 0.2M calcium chloride solution were added to different sample cups, respectively, and then 340 μL of samples with different detection ranges (quality control) were added, respectively, and mixed for 3 times, and the detection was started.

[0091] Using different amounts of coagulation activation detection reagent (8 μL, 10 μL, 12 μL), different detection ranges of samples (normal human samples, coagulation activation quality control level 1, coagulation activation quality control level 2, 20220101 batch comparative quality control, 20220614 batch comparative quality control) were detected, and the corresponding TEG-ACT values were obtained. Taking 10 μL of coagulation activation detection reagent as a reference, the average relative deviation of 8 μL and 12 μL detection results is ≥5%, then the detection sample has greater sensitivity to the coagulation activation detection reagent, and the detection sample can be used for sensitivity evaluation of the coagulation activation detection reagent; otherwise, the detection sample is not suitable for sensitivity evaluation of the coagulation activation detection reagent. The experimental results are shown in Table 6. The detection map of one of the 8 μL detection results of the normal human sample is shown in Figure 11 ; the detection map of one of the 8 μL detection results of the 20220301 batch coagulation activation quality control level 1 is shown in Figure 12 ; the detection map of one of the 8 μL detection results of the 20220301 batch coagulation activation quality control level 2 is shown in Figure 13 ; the detection map of one of the 8 μL detection results of the 20220101 batch comparative quality control is shown in Figure 14 ; the detection map of the 8 μL result 1 of the 20220614 batch comparative quality control is shown in Figure 15 ”.

[0092] The evaluation results show that the TEG-ACT values of normal human sample, coagulation activation control level 1, and 20220101 batch of comparative control are small (< 130 S), the sensitivity of the detection sample to the coagulation activation detection reagent is low, and the abnormal conditions (such as content change, failure, etc.) of the coagulation activation detection reagent cannot be effectively evaluated; while the TEG-ACT values of coagulation activation control level 2 and 20220614 batch of comparative control are large (≥ 130 S), the sensitivity of the detection sample to the coagulation activation detection reagent is high, and the abnormal conditions (such as content change, failure, etc.) of the coagulation activation detection reagent can be effectively evaluated, but the TEG-ACT value of 20220614 batch of comparative control is too large (≥ 200 S), and the stability is poor (detection method, see “3. Performance detection of coagulation activation control level 1”). Therefore, the TEG-ACT value of the sample is controlled at 160 ± 30 S, and the coagulation activation control level 2 can effectively evaluate the abnormal conditions (such as content change, failure, etc.) of the coagulation activation detection reagent, and also ensure the stability of the sample.

[0093] Table 6: Sensitivity detection results of samples with different detection value ranges to coagulation activation detection reagent

[0094]

[0095] Note: Mean relative deviation = (8 μL or 12 μL TEG-ACT mean value - 10 μL TEG-ACT mean value) / 10 μL TEG-ACT mean value x 100%.

[0096] The thrombus activation quality control level 1 and the thrombus activation quality control level 2 can be used to evaluate the stability and accuracy of the rapid activation detection system composed of the thrombelastograph and the thrombelastograph test (thrombus activation) reagent (coagulation method). According to the thrombelastograph test (thrombus activation) reagent (coagulation method) instruction, the detection results such as TEG-ACT value and MA value fall within the assigned range, proving that the rapid activation detection system can be used for clinical sample detection. Otherwise, the thrombelastograph needs to be debugged or it is necessary to confirm whether the reagent is invalid. Among them, level 2 is more sensitive to the reagent. In the case that the thrombelastograph is not faulty, the amount of effective components of the reagent is too high or too low, which will cause the TEG-ACT value of the quality control level 2 to exceed its assigned range. It is proved that level 2 is more suitable for evaluating whether the thrombus activation detection reagent is abnormal (such as low content, invalidity, etc.). That is, in the preparation process, the quality control product with TEG-ACT value of 160±30S is formed, which can be used to effectively evaluate the abnormal condition of the reagent. The TEG-ACT value of the normal human sample or level 1 is not within the above range, and it is not suitable as a quality control product for evaluating the abnormal condition of the reagent. In the prior art, there is a lack of quality control product for the rapid activation detection system of the present scheme, and the normal human sample is usually used instead, which has the problems of poor stability and poor sensitivity to abnormal conditions of the thrombus activation reagent.

[0097] In summary, the thrombus activation quality control level 1 and the thrombus activation quality control level 2 have good stability, can be used to evaluate the stability and accuracy of the rapid activation detection system, and simulate the state of normal coagulation and abnormal coagulation, respectively. In addition to the above functions, the thrombus activation quality control level 2 can be used to detect whether the thrombus activation reagent (coagulation method) is abnormal.

[0098] The above is only an embodiment of the present application, and the specific technical solutions and / or common knowledge of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the technical solutions of the present application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope claimed in this application should be subject to the content of its claims, and the specific embodiments described in the specification can be used to explain the content of the claims.

Claims

1. Two-level coagulation activation detection reagent quality control product, characterized by: These include coagulation activation quality control level 1 with a TEG-ACT value of 100 ± 20 s and a MA value of 60 ± 15 mm, and coagulation activation quality control level 2 with a TEG-ACT value of 160 ± 30 s; The raw materials of coagulation activation quality control product level 1 include plasma matrix fluid, fibrinogen matrix fluid, anticoagulant, pH buffer, protein protectant and preservative; The raw materials of coagulation activation quality control products at level 2 include plasma matrix, anticoagulant, pH buffer, protein protectant and preservative; The anticoagulant is at least one of bivalirudin, edoxaban and enoxaparin sodium; Coagulation Activation Quality Control Level 1 and Coagulation Activation Quality Control Level 2 are used to evaluate the stability and accuracy of the coagulation activation detection system consisting of a thrombelastograph and coagulation activation detection reagents; Coagulation Activation Control Level 2 is used to evaluate the stability and accuracy of coagulation activation assays.

2. The two-level coagulation activation detection reagent quality control product according to claim 1, characterized in that: The protein protective agent includes at least one of bovine serum albumin, sucrose, trehalose, glycine, mannitol and lactose; the pH buffer is at least one of Hepes buffer, Tris-HCl buffer and phosphate buffer; and the preservative is at least one of Proclin 300, sodium azide and sodium benzoate.

3. The two-level coagulation activation detection reagent quality control product according to claim 1, characterized in that: The plasma matrix liquid and the fibrinogen matrix liquid are prepared by the following method: sodium citrate is added to fresh pig whole blood to obtain anticoagulated pig blood; the anticoagulated pig blood is allowed to stand for stratification, and the upper layer of liquid is taken, and then centrifuged to obtain the supernatant to obtain pig plasma; after the pig plasma is frozen and thawed and centrifuged, the upper layer is taken to obtain plasma matrix liquid, and the lower layer is taken to obtain fibrinogen matrix liquid.

4. The method for preparing a quality control product of a two-level coagulation activation detection reagent according to any one of claims 1 to 3, characterized in that: The method includes the following steps: S1: mixing plasma matrix solution, fibrinogen matrix solution, anticoagulant, pH buffer, protein protectant and preservative to obtain a stock solution; S2: Adjust the TEG-ACT value and / or MA value of the stock solution to the specified range to obtain the stock solution to be lyophilized, and then obtain the quality control product through lyophilization.

5. The method for preparing a quality control product of a two-level coagulation activation detection reagent according to claim 4, characterized in that: In S2, the TEG-ACT value of the stock solution was adjusted to 100 ± 20S and the MA value was 60 ± 15 mm to obtain a coagulation activation quality control level of 1.

6. The method for preparing a quality control product of a two-level coagulation activation detection reagent according to claim 4, characterized in that: In S2, the TEG-ACT value of the stock solution was adjusted to 160 ± 30S to obtain a coagulation activation quality control level 2.

7. Use of the two-level coagulation activation detection reagent quality control product according to any one of claims 1 to 3 in evaluating the stability and accuracy of a coagulation activation detection system, characterized in that: The coagulation activation detection system includes a thrombelastograph and a coagulation activation detection reagent.

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