阅读说明：本技术 一种血栓弹力图法增强型纤溶系统检测试剂盒及其制备方法 (Thromboelastography enhanced fibrinolysis system detection kit and preparation method thereof ) 是由 王连升 李红梅 于 2020-06-02 设计创作，主要内容包括：本发明提供了一种血栓弹力图法增强型纤溶系统检测试剂盒,属于检测技术领域。该试剂盒包括以下组分：pH值为7.2～7.4的4-羟乙基哌嗪乙磺酸缓冲液、高岭土、多聚赖氨酸、甘氨酸、牛血清白蛋白、纤溶酶原激活物、海藻糖、聚蔗糖、PEG8000,脑磷脂、NaCl、KCl、纳豆激酶和Proclin300。试剂盒中包括纤溶酶原激活物,纳豆激酶等,将纤溶检测时间缩短为30min内,能快速区分原发和继发纤溶亢进、早期诊断DIC,满足临床时效性需求。(The invention provides a thromboelastography enhanced fibrinolysis system detection kit, and belongs to the technical field of detection. The kit comprises the following components: 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution with the pH value of 7.2-7.4, kaolin, polylysine, glycine, bovine serum albumin, plasminogen activator, trehalose, polysucrose, PEG8000, cephalin, NaCl, KCl, nattokinase and Proclin 300. The kit comprises plasminogen activator, nattokinase and the like, shortens the fibrinolysis detection time to be within 30min, can quickly distinguish primary and secondary hyperfibrino and diagnose DIC at early stage, and meets the clinical timeliness requirement.)

1. A thromboelastography enhanced fibrinolysis system detection kit is characterized by comprising the following components: 5-100 mmol/L HEPES buffer solution with the pH value of 7.2-7.4, 0.1-0.6 g/L kaolin, 5-30 g/L polylysine, 10-60 g/L glycine, 5-30 g/L BSA, 5 IU-1000 IU/mL plasminogen activator, 5-30 g/L trehalose, 1-12 g/L polysucrose, 1-20 g/L PEG8000, 10-60 mg/L cephalin, 6-12g/L NaCl, 4-8 g/L KCl, 10-1000 IU/mL nattokinase, and 0.03-0.05% Proclin 300.

2. The thromboelastography-enhanced fibrinolysis system test kit according to claim 1, wherein the activity of said plasminogen activator is 5000 IU/g; the activity of the nattokinase is 40000 IU/g.

3. The thromboelastography-enhanced fibrinolysis system detection kit according to claim 1, characterized by comprising the following components: 10mmol/L HEPES buffer solution with pH value of 7.2, 0.5g/L kaolin, 20g/L polylysine, 40g/L glycine, 20g/L BSA, 50IU/mL plasminogen activator, 20g/L trehalose, 8g/L ficoll, 10g/L PEG8000, 50mg/L cephalin, 9g/L NaCl, 6g/L KCl, 800IU/mL natto kinase, and 0.05% Proclin 300.

4. The thromboelastography-enhanced fibrinolysis system detection kit according to claim 1, characterized by comprising the following components: 50mmol/L HEPES buffer solution with pH value of 7.3, 0.25g/L kaolin, 30g/L polylysine, 20g/L glycine, 30g/L BSA, 250IU/mL plasminogen activator, 10g/L trehalose, 12g/L ficoll, 20g/L PEG8000, 25mg/L cephalin, 6g/L NaCl, 8g/L KCl, 400IU/mL natto kinase, and 0.04% Proclin 300.

5. The thromboelastography-enhanced fibrinolysis system detection kit according to claim 1, characterized by comprising the following components: 100mmol/L HEPES buffer solution with pH value of 7.4, 0.1g/L kaolin, 10g/L polylysine, 60g/L glycine, 5g/L BSA, 800IU/mL plasminogen activator, 30g/L trehalose, 2g/L polysucrose, 5g/L PEG8000, 10mg/L cephalin, 12g/L NaCl, 4g/L KCl, 100IU/mL natto kinase and 0.03% Proclin 300.

6. A method for preparing the thromboelastography-enhanced fibrinolysis system detection kit as claimed in any one of claims 1 to 5, comprising the following steps:

s1, adding 4-hydroxyethyl piperazine ethanesulfonic acid into purified water to prepare a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, and adjusting the pH value of the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution to 7.2-7.4;

s2, adding kaolin into purified water to prepare a kaolin stock solution;

s3, adding the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution prepared in the step S1 into the cerebral jelly, and grinding the mixture to be milky to prepare a cerebral jelly stock solution;

s4, adding purified water into the plasminogen activator freeze-dried powder for redissolution to prepare plasminogen activator stock solution;

s5, adding polylysine, glycine, PEG8000, polysucrose, nattokinase, trehalose, bovine serum albumin, NaCl and KCl into the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution prepared by S1 to prepare a solution;

and S6, adding the kaolin stock solution prepared in the S2, the brain gel stock solution prepared in the S3 and the plasminogen activator stock solution prepared in the Proclin300 and the S4 into the solution prepared in the S5, adding the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution prepared in the S1, uniformly mixing, and freeze-drying to obtain the thromboelastography enhanced fibrinolysis system detection kit.

7. The method for preparing the thromboelastography-enhanced fibrinolysis system detection kit according to claim 6, further comprising a subpackaging step of subpackaging 20ul of each reagent bottle and freeze-drying to obtain the thromboelastography-enhanced fibrinolysis system detection kit.

8. The method for preparing the thromboelastography-enhanced fibrinolysis system detection kit according to claim 6, wherein the concentration of the 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution prepared in S1 is 10mmol/L, and the pH value is 7.2; the concentration of the kaolin stock solution prepared in the S2 is 5 g/L; the concentration of the brain gel stock solution prepared in the S3 is 1 g/L; the concentration of the plasminogen activator stock solution prepared in the S4 is 5000 IU/mL; weighing 0.2g of polylysine, 0.4g of glycine, 0.1g of PEG8000, 0.08g of polysucrose, 0.2g of nattokinase with the activity of 40000IU/g, 0.2g of trehalose, 0.2g of BSA, 0.09g of NaCl and 0.06g of KCl in the solution prepared in the S5; in 10mL of the solution prepared in the S6, 0.5g/L of kaolin, 50mg/L of encephalic coagulum, 50IU/mL of plasminogen activator, 0.05% of Proclin, 20g/L of polylysine, 40g/L of glycine, 800010g/L of PEG, 8g/L of polysucrose, 800IU/mL of nattokinase, 20g/L of trehalose, 20g/L of BSA, 9g/L of NaCl and 6g/L of KCl are contained.

9. The method for preparing the thromboelastography-enhanced fibrinolysis system detection kit according to claim 7, wherein the concentration of the 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution prepared in S1 is 50mmol/L, and the pH value is 7.3; the concentration of the kaolin stock solution prepared in the S2 is 5 g/L; the concentration of the brain gel stock solution prepared in the S3 is 1 g/L; the concentration of the plasminogen activator stock solution prepared in the S4 is 5000 IU/mL; weighing 0.3g of polylysine, 0.2g of glycine, 0.2g of PEG8000, 0.12g of polysucrose, 0.1g of nattokinase with the activity of 40000IU/g, 0.1g of trehalose, 0.3g of BSA, 0.06g of NaCl and 0.08g of KCl in the solution prepared in the S5; 0.25g/L of kaolin, 25mg/L of encephalin, 250IU/mL of plasminogen activator, 0.04% of Proclin, 30g/L of polylysine, 20g/L of glycine, 800020g/L of PEG, 12g/L of polysucrose, 400IU/mL of nattokinase, 10g/L of trehalose, 30g/L of BSA, 6g/L of NaCl and 8g/L of KCl in 10mL of solution prepared in the S6.

10. The method for preparing the thromboelastography-enhanced fibrinolysis system detection kit according to claim 7, wherein the concentration of the 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution prepared in S1 is 100mmol/L, and the pH value is 7.4; the concentration of the kaolin stock solution prepared in the S2 is 5 g/L; the concentration of the brain gel stock solution prepared in the S3 is 1 g/L; the concentration of the plasminogen activator stock solution prepared in the S4 is 5000 IU/mL; weighing 0.1g of polylysine, 0.6g of glycine, 0.05g of PEG8000, 0.02g of polysucrose, 0.025g of nattokinase with the activity of 40000IU/g, 0.3g of trehalose, 0.05g of BSA, 0.12g of NaCl and 0.04g of KCl in the solution prepared in the S5; in 10mL of the solution prepared in the S6, 0.1g/L of kaolin, 10mg/L of encephalin, 800IU/mL of plasminogen activator, 0.03% of Proclin, 10g/L of polylysine, 60g/L of glycine, 80005g/L of PEG80005, 2g/L of polysucrose, 100IU/mL of nattokinase, 30g/L of trehalose, 5g/L of BSA, 12g/L of NaCl and 4g/L of KCl are contained.

Technical Field

The invention relates to the technical field of detection, in particular to a thromboelastography enhanced fibrinolysis system detection kit and a preparation method thereof.

Background

A dynamic equilibrium relationship exists among the fibrinolytic system, the blood coagulation system and the platelets in the human body under normal physiological conditions, so that the stability and smoothness of blood circulation are guaranteed. The fibrinolytic system functions to remove fibrin deposited on blood vessels, dissolve blood clots, maintain a smooth blood flow, prevent thrombosis and eliminate formed thrombus.

The process of decomposing and liquefying fibrin formed in the blood coagulation process is called fibrinolysis, which is called fibrinolysis for short. An abnormally enhanced fibrinolytic activity, i.e., hyperfibrinolysis. Hyperfibrinolysis is classified into primary hyperfibrinolysis and secondary hyperfibrinolysis, which can cause severe complications such as hemorrhage.

Primary fibrinolysis is a clinical manifestation characterized by skin bleeding such as massive plaque and mucosal visceral bleeding due to increased plasmin activity caused by increased plasminogen activator, which degrades fibrinogen and various coagulation factors in plasma, decreasing their plasma levels and activity.

Secondary fibrinolysis refers to hyperfibrinolysis secondary to intravascular coagulation, and is mainly seen in Disseminated Intravascular Coagulation (DIC). DIC is a pathological process characterized by abnormal blood coagulation function, which is caused by activation of coagulation factors and platelets by some pathogenic factors and invasion of a large amount of soluble procoagulant substances into the blood. A large amount of microthrombosis are formed in microcirculation, blood coagulation factors and blood platelets are consumed in a large amount, and a secondary fibrinolysis process is enhanced, so that clinical manifestations such as bleeding, shock, organ dysfunction, anemia and the like appear. Causing multiple organ dysfunction syndrome in patients with DIC that has been initiated will be the leading cause of death. DIC fatality rates as high as 31% -80%, its clinical diagnosis is relatively difficult, and different patients have different treatment decisions. DIC is a complex pathophysiological process in which inflammation and the coagulation system interact with each other. Thrombelastogram was invented by Hellmut Hartert, a German scientist, in 1948 and was widely used in the clinic since the 1980 s. The thromboelastogram can more accurately reflect the whole process of blood coagulation, can diagnose high coagulation and low coagulation more early, the low coagulation is closely related to poor prognosis of sepsis, and a good effect is obtained on blood transfusion in a guiding operation, so that the thromboelastogram is the most important index for monitoring the blood coagulation function in the perioperative period.

Compared with the traditional blood coagulation detection, the thromboelastogram uses whole blood as a detection sample, adds an activator in vitro to start a blood coagulation mechanism, monitors the whole process from the start of an internal and external blood coagulation system, the formation of fibrin to the dissolution of blood clots, can more accurately, more intuitively and more comprehensively reflect the comprehensive state of all blood coagulation factors except vascular endothelial cells and vascular walls in the blood coagulation mechanism, and has very definite guiding significance for clinical treatment.

The detection process of the thrombelastogram (shown in figure 1) is that a test cup loaded with a blood specimen rotates back and forth at a constant speed at a specific angle, once a blood clot forms, a metal torsion wire connected with a piston in the test cup is subjected to the shear stress formed by the blood clot, and then rotates left and right, and the thrombelastogram is obtained according to the drawing of the rotating amplitude of the metal torsion wire. Thromboelastography enables dynamic monitoring of the coagulation, fibrinolysis and functional detection of platelets throughout the coagulation process and guidance of component transfusion.

The main parameters of the thromboelastogram include R, K, alpha angle, MA, LY30, EPL, LY60 and the like, wherein LY30, EPL and LY60 are indexes for measuring fibrinolysis, and different parameters represent the following meanings:

LY 30: the rate (%) of clot minute lump ablation (or reduction) within 30 minutes after MA value determination was measured, reflecting the fibrinolytic activity of blood 30 minutes after MA value determination. LY30 > 7.5%, which indicates a high fibrinolysis state, i.e. hyperfibrinolysis. LY30 > 7.5%, if CI is less than or equal to 1.0, indicating primary hyperfibrino lysis, correcting by using anti-fibrinolytic drugs; if CI is more than or equal to 3.0, the hyperfibrinolysis is prompted, and anticoagulation treatment is required.

CI: a coagulation complex index for describing the overall coagulation status of a patient < 3: low freezing, -3 < normal +3, with a value of + 3: high coagulation. This parameter has considerable implications for the prediction of thrombosis and hemorrhage.

EPL: the percentage of blood clot that will dissolve within 30 minutes after MA value determination is predicted. EPL ═ 100(MA-a 30)/MA.

LY 60: the rate of decrease of the amplitude of the clot within 60 minutes after determination of the MA value is measured, LY60 > 15%, indicating a hyperfibrinolytic state, i.e. hyperfibrinolysis.

R value: the time required from the start of the placement of the blood sample in the test cup to the formation of the first fibrin clot (trace amplitude up to 2mm) reflects the combined effect of all coagulation factors involved in the coagulation process (intrinsic, extrinsic and common pathways). The R value can be prolonged by the lack of anticoagulant and coagulation factor, and shortened by the high coagulation state of blood.

K value: the time from the end of the R time to the trace amplitude of 20mm reflects the result of the co-action of fibrin and platelets at the onset of clot formation, reflecting the rate of clot formation. The length of K is mainly influenced by the level of fibrinogen, but is less influenced by platelet function. Anticoagulants can prolong the K value.

Angle α: the angle between the tangent and the horizontal line from the point of clot formation to the maximum curve arc of the trace is closely related to the value of K, both reflecting the rate of clot aggregation. When the specimen is in a severe low coagulation state, the maximum amplitude of the blood clot can not reach 20mm, and the K value can not be determined. Thus, the α angle is more valuable than the K value. The factor affecting the angle alpha is the same as the value of K.

MA: the maximum amplitude of the thrombelastogram, i.e., the maximum shear stress coefficient (mm). Reflecting the maximum strength of the clot and the stability of clot formation, reflects the combined effects of fibrin level and platelet function.

At present, the MA value is determined in about 35 minutes in the conventional test of the thromboelastography, and the fibrinolysis test is continued for 30min after the MA value is determined, so that the fibrinolysis test time is at least 60min, and the clinical timeliness requirement cannot be met.

Disclosure of Invention

In order to solve the technical problem that the fibrinolysis test time in the prior art cannot meet the clinical timeliness requirement, the invention provides a thrombus elastography enhanced fibrinolysis system detection kit, and therefore, the invention also provides a preparation method of the thrombus elastography enhanced fibrinolysis system detection kit.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention aims to provide a thromboelastography enhanced fibrinolysis system detection kit, which comprises the following components: 5-100 mmol/L HEPES buffer solution with the pH value of 7.2-7.4, 0.1-0.6 g/L kaolin, 5-30 g/L polylysine, 10-60 g/L glycine, 5-30 g/L BSA, 5 IU-1000 IU/mL plasminogen activator, 5-30 g/L trehalose, 1-12 g/L polysucrose, 1-20 g/L PEG8000, 10-60 mg/L cephalin, 6-12g/L NaCl, 4-8 g/L KCl, 10-1000 IU/mL nattokinase and 0.03-0.05% Proclin 300.

Preferably, the plasminogen activator activity is 5000 IU/g; the activity of the nattokinase is 40000 IU/g.

Preferably, the following components are included: 10mmol/L HEPES buffer solution with pH value of 7.2, 0.5g/L kaolin, 20g/L polylysine, 40g/L glycine, 20g/L BSA, 50IU/mL plasminogen activator, 20g/L trehalose, 8g/L ficoll, 10g/L PEG8000, 50mg/L cephalin, 9g/L NaCl, 6g/L KCl, 800IU/mL natto kinase, and 0.05% Proclin 300.

Preferably, the following components are included: 50mmol/L HEPES buffer solution with pH value of 7.3, 0.25g/L kaolin, 30g/L polylysine, 20g/L glycine, 30g/L BSA, 250IU/mL plasminogen activator, 10g/L trehalose, 12g/L ficoll, 20g/L PEG8000, 25mg/L cephalin, 6g/L NaCl, 8g/L KCl, 400IU/mL natto kinase, and 0.04% Proclin 300.

Preferably, the following components are included: 100mmol/L HEPES buffer solution with pH value of 7.4, 0.1g/L kaolin, 10g/L polylysine, 60g/L glycine, 5g/L BSA, 800IU/mL plasminogen activator, 30g/L trehalose, 2g/L polysucrose, 5g/L PEG8000, 10mg/L cephalin, 12g/L NaCl, 4g/L KCl, 100IU/mL natto kinase and 0.03% Proclin 300.

The invention also aims to provide a preparation method of the thrombus elastography enhanced fibrinolysis system detection kit, which comprises the following steps:

s1, adding 4-hydroxyethyl piperazine ethanesulfonic acid into purified water to prepare a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, and adjusting the pH value of the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution to 7.2-7.4;

s2, adding kaolin into purified water to prepare a kaolin stock solution;

s3, adding the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution prepared in the step S1 into the cerebral jelly, and grinding the mixture to be milky to prepare a cerebral jelly stock solution;

s4, adding purified water into the plasminogen activator freeze-dried powder for redissolution to prepare plasminogen activator stock solution;

s5, adding polylysine, glycine, PEG8000, polysucrose, nattokinase, trehalose, bovine serum albumin, NaCl and KCl into the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution prepared by S1 to prepare a solution;

and S6, adding the kaolin stock solution prepared in the S2, the brain gel stock solution prepared in the S3 and the plasminogen activator stock solution prepared in the Proclin300 and the S4 into the solution prepared in the S5, adding the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution prepared in the S1, uniformly mixing, and freeze-drying to obtain the thromboelastography enhanced fibrinolysis system detection kit.

Preferably, the kit further comprises a subpackaging step, wherein each reagent bottle is subpackaged with 20ul, and the thromboelastography enhanced fibrinolysis system detection kit is obtained by freeze-drying.

Preferably, the concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution prepared in the S1 is 10mmol/L, and the pH value is 7.2; the concentration of the kaolin stock solution prepared in the S2 is 5 g/L; the concentration of the brain gel stock solution prepared in the S3 is 1 g/L; the concentration of the plasminogen activator stock solution prepared in the S4 is 5000 IU/mL; weighing 0.2g of polylysine, 0.4g of glycine, 0.1g of PEG8000, 0.08g of polysucrose, 0.2g of nattokinase with the activity of 40000IU/g, 0.2g of trehalose, 0.2g of BSA, 0.09g of NaCl and 0.06g of KCl in the solution prepared in the S5; in 10mL of the solution prepared in the S6, 0.5g/L of kaolin, 50mg/L of encephalic coagulum, 50IU/mL of plasminogen activator, 0.05% of Proclin, 20g/L of polylysine, 40g/L of glycine, 800010g/L of PEG, 8g/L of polysucrose, 800IU/mL of nattokinase, 20g/L of trehalose, 20g/L of BSA, 9g/L of NaCl and 6g/L of KCl are contained.

Preferably, the concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution prepared in the S1 is 50mmol/L, and the pH value is 7.3; the concentration of the kaolin stock solution prepared in the S2 is 5 g/L; the concentration of the brain gel stock solution prepared in the S3 is 1 g/L; the concentration of the plasminogen activator stock solution prepared in the S4 is 5000 IU/mL; weighing 0.3g of polylysine, 0.2g of glycine, 0.2g of PEG8000, 0.12g of polysucrose, 0.1g of nattokinase with the activity of 40000IU/g, 0.1g of trehalose, 0.3g of BSA, 0.06g of NaCl and 0.08g of KCl in the solution prepared in the S5; 0.25g/L of kaolin, 25mg/L of encephalin, 250IU/mL of plasminogen activator, 0.04% of Proclin, 30g/L of polylysine, 20g/L of glycine, 800020g/L of PEG, 12g/L of polysucrose, 400IU/mL of nattokinase, 10g/L of trehalose, 30g/L of BSA, 6g/L of NaCl and 8g/L of KCl in 10mL of solution prepared in the S6.

Preferably, the concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution prepared in the S1 is 100mmol/L, and the pH value is 7.4; the concentration of the kaolin stock solution prepared in the S2 is 5 g/L; the concentration of the brain gel stock solution prepared in the S3 is 1 g/L; the concentration of the plasminogen activator stock solution prepared in the S4 is 5000 IU/mL; weighing 0.1g of polylysine, 0.6g of glycine, 0.05g of PEG8000, 0.02g of polysucrose, 0.025g of nattokinase with the activity of 40000IU/g, 0.3g of trehalose, 0.05g of BSA, 0.12g of NaCl and 0.04g of KCl in the solution prepared in the S5; in 10mL of the solution prepared in the S6, 0.1g/L of kaolin, 10mg/L of encephalin, 800IU/mL of plasminogen activator, 0.03% of Proclin, 10g/L of polylysine, 60g/L of glycine, 80005g/L of PEG80005, 2g/L of polysucrose, 100IU/mL of nattokinase, 30g/L of trehalose, 5g/L of BSA, 12g/L of NaCl and 4g/L of KCl are contained.

The thromboelastography enhanced fibrinolysis system detection kit provided by the invention comprises plasminogen activator, nattokinase and the like, shortens the fibrinolysis detection time to be within 30min, can quickly distinguish primary and secondary hyperfibrino and early diagnosis DIC, and meets the clinical timeliness requirement.

The invention provides a preparation method of a thromboelastography enhanced fibrinolysis system detection kit, which is characterized in that plasminogen activator and nattokinase are added, and the fibrinolysis process comprises two parts, namely plasminogen activation and fibrin or fibrinogen degradation. Plasmin has two intrinsic and extrinsic activation pathways. Plasminogen activator belongs to endogenous activator, natto kinase belongs to endogenous and exogenous activators, and the two are mutually synergistic to shorten the fibrinolysis detection time to be within 30 min.

Drawings

FIG. 1 is a schematic diagram of thrombus elasticity (left: detection principle; right: result map analysis)

FIG. 2. Curve 1 shows the conventional fibrinolysis assay, and Curve 2 shows the fibrinolysis assay of the present invention

FIG. 3 is a graph showing the effect of the tests of comparative examples 1 to 3 on the scale of examples 1 to 3.

Detailed Description

The technical scheme provided by the invention is further explained in detail by combining a specific embodiment;

kaolin, polylysine, glycine, bovine serum albumin, plasminogen activator, trehalose, sucrose, PEG8000, cephalin, NaCl, KCl, nattokinase, Proclin300, 4-hydroxyethylpiperazine ethanesulfonic acid, and other reagents according to the following examples were commercially available.

HEPES is 4-hydroxyethyl piperazine ethanesulfonic acid; BSA is bovine serum albumin; proclin300 is a commercially available preservative; PEG8000 is polyethylene glycol; kaolin is Kaolin.