Thromboelastography activated coagulation detection reagent and its application

Abstract

The present invention relates to the technical field of in vitro coagulation detection, and more specifically to a thromboelastography activated coagulation detection reagent and its application. The activated coagulation detection reagent provided by the present invention utilizes nano-sized powdered kaolin in combination with cephalin to effectively activate the endogenous coagulation activation pathway. The three-dimensional network structure formed by gellan gum and divalent metal magnesium ions has excellent support and extremely low viscosity, enabling the reagent to maintain good fluidity and suspension ability, reducing precipitation of the active ingredient, and effectively preventing the activated coagulation detection reagent from becoming ineffective during long-term storage and transportation.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro coagulation detection, and in particular to a thromboelastogram activated coagulation detection reagent and application thereof. Background Art

[0002] The thromboelastograph (TEG) is a specialized graph produced by a thromboelastometer that accurately and comprehensively assesses blood coagulation in vitro. TEG provides scientific, objective, and reliable data for guiding component transfusions, assessing the efficacy of procoagulant and anticoagulant medications, assessing the risk of thrombosis in patients, and preventing postoperative thrombosis.

[0003] German professor Harter first described thromboelastometry in 1948. Starting in 2000, thromboelastometry began to be introduced in my country's tertiary hospitals, with use taking the lead in departments like anesthesiology, ICU, extracorporeal circulation, and organ transplantation. By June 2007, thromboelastometry was included in the "Catalogue of Clinical Laboratory Items for Medical Institutions" and the "National Medical Service Price Item Specifications (Trial 2001 Edition)." The stainless steel blood cup in the thrombelastograph rotates back and forth at an angle of 4°45'. When the blood sample between the cup wall and the cylinder in the cup is liquid, the back-and-forth rotation of the cup cannot move the cylinder, and the signal reflected by the sensor on the tracing paper is a straight line. When the blood begins to coagulate, resistance is generated between the cup and the cylinder due to the adhesion of fibrin. The rotation of the cup drives the cylinder to move at the same time. As the fibrin increases, the resistance also continues to increase, and the movement of the cylinder driven by the cup also changes accordingly. The movement of the cylinder cuts the magnetic lines of force to generate current, which is converted into a digital signal. This signal is depicted on the tracing paper through the sensor to form a unique thrombelastograph.

[0004] Currently, most fully automated thromboelastography activated coagulation test reagents are packaged in large bottles. However, due to the long testing time, the activator kaolin can partially settle during the test, resulting in uneven test reagents and affecting test results. For example, an existing fully automated thromboelastography activated coagulation test reagent uses activators including kaolin, phospholipids, and sodium azide. While this reduces costs and provides good short-term stability, it cannot guarantee long-term stability or reagent uniformity, and it also cannot prevent kaolin precipitation. Therefore, there is an urgent need to develop a fully automated thromboelastography activated coagulation test reagent that is stable, non-precipitating, and uniform. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a thromboelastography activated coagulation detection reagent and its application. The present invention provides an activated coagulation detection reagent that can keep kaolin suspended for a long time and avoid precipitation of effective reagents, and its application in thromboelastography tests.

[0006] The present invention provides an activated coagulation detection reagent, comprising 0.1% to 0.6% (w / v) Tris, 0.01% to 0.05% (w / v) kaolin, 0.1% to 2% (v / v) cephalin, 0.1% to 0.5% (w / v) stabilizer, 0.01% to 2% (w / v) metal ion and 0.05% (w / v) preservative;

[0007] W / v is the mass-to-volume ratio, in g / mL;

[0008] The stabilizer is selected from at least one of gellan gum, agar, carrageenan, and xanthan gum;

[0009] The metal ion is selected from Mg 2+ 、Zn 2+ 、Mn 2+ At least one of them.

[0010] Furthermore, the activated coagulation detection reagent provided by the present invention has good stability, is not prone to precipitation, and has good uniformity. The activated coagulation detection reagent is used in conjunction with a thromboelastograph to detect relevant indicators.

[0011] In some embodiments, the present invention screens the components and concentrations of activated coagulation detection reagents by designing experiments. The results show that the components of the activated coagulation detection reagent are Tris, kaolin, cephalin, stabilizers, metal ions and preservatives. The detection effect is better when the concentration is 0.1% to 0.6% (w / v) Tris, 0.01% to 0.05% (w / v) kaolin, 0.1% to 2% (v / v) cephalin, 0.1% to 0.5% (w / v) stabilizer, 0.01% to 2% (w / v) metal ions and 0.05% (w / v) preservative.

[0012] Specifically, the activated coagulation detection reagent provided by the present invention includes 0.3% (w / v) Tris, 0.035% (w / v) kaolin, 1% (v / v) cephalin, 0.2% (w / v) stabilizer, 0.15% (w / v) metal ion and 0.05% (w / v) preservative.

[0013] In the activated coagulation detection reagent provided by the present invention, the stabilizer is gellan gum, and the metal ion is Mg 2+ .

[0014] Specifically, the stabilizer includes gellan gum, agar, carrageenan, xanthan gum, sodium alginate, gum arabic, pectin, guar gum, locust bean gum, gelatin, and Tween. The present invention proves through experiments that low acyl gellan gum can form a special gel with divalent metal ions by relying on its free groups, so it can be combined with an appropriate amount of Mg 2+The combination can form a three-dimensional network structure, which has good supporting capacity, pseudoplasticity and extremely low viscosity, so that the reagent maintains good fluidity and suspension ability.

[0015] The pH value of the activated coagulation detection reagent provided by the present invention is 7.0-8.5.

[0016] Specifically, in some embodiments, the pH value of the activated coagulation detection reagent is 7.3.

[0017] In the activated coagulation detection reagent provided by the present invention, the metal ions are specifically:

[0018] The Mg 2+ From at least one of MgCl2, MgSO4, and Mg(NO3)2;

[0019] The Zn 2+ From at least one of ZnCl2, ZnSO4, and Zn(NO3)2;

[0020] The Mn 2+ It is derived from at least one of MnCl2, MnSO4, and Mn(NO3)2, but the present invention does not impose any limitation thereto.

[0021] In the activated coagulation detection reagent provided by the present invention, the kaolin is nano-grade powdered kaolin.

[0022] Specifically, the endogenous coagulation activator includes kaolin, ellagic acid, and diatomaceous earth. The nano-sized powdered kaolin used in the present invention, when combined with gellan gum, allows the fine kaolin particles to be suspended in the medium for a long time, thereby improving the stability and uniformity of the reagent and preventing precipitation of the active ingredients in the reagent.

[0023] In the activated coagulation detection reagent provided by the present invention, the preservative is gentamicin sulfate.

[0024] Specifically, the preservatives include sodium azide, thimerosal, kasone, gentamicin, and Proclin 300. The gentamicin sulfate used in the present invention is an aminoglycoside drug that has strong antibacterial activity against Pseudomonas aeruginosa, Proteus, Escherichia coli, Klebsiella, Enterobacter, Serratia, Shigella, Citrobacter, Neisseria, and Staphylococcus aureus.

[0025] The activated coagulation detection reagent provided by the present invention specifically includes water, 0.3% (w / v) Tris, 0.035% (w / v) kaolin, 1% (v / v) cephalin, 0.2% (w / v) gellan gum, 0.15% (w / v) magnesium chloride and 0.05% (w / v) gentamicin sulfate.

[0026] Specifically, in some embodiments, when the components of the activated coagulation detection reagent are water, 0.3% (w / v) Tris, 0.035% (w / v) kaolin, 1% (w / v) cephalin, 0.2% (w / v) gellan gum, 0.15% (w / v) magnesium chloride and 0.05% (w / v) gentamicin sulfate, the detection results of blood samples are better than those of activated coagulation detection reagents with other components, and the stability is higher and the uniformity is better.

[0027] The present invention also provides the use of the activated coagulation detection reagent in preparing a coagulation method activated coagulation detection kit.

[0028] The present invention also provides a coagulation method activated coagulation detection kit, which comprises the activated coagulation detection reagent and calcium chloride. The concentration of the calcium chloride is 0.1-1 mol / L, preferably 0.2 mol / L.

[0029] In the solution provided by the present invention, kaolin and cephalin can effectively start the endogenous coagulation activation pathway, thereby performing accurate thromboelastography detection. 2+ The two stabilizers combine to form a three-dimensional network structure, which has good supporting capacity, pseudoplasticity and extremely low viscosity, enabling the reagent to maintain good fluidity and suspension ability, significantly improving the stability of the activated coagulation detection reagent, and effectively avoiding the problem of failure of the activator during long-term storage and transportation. DETAILED DESCRIPTION

[0030] The present invention provides thromboelastography activated coagulation detection reagents and their applications. Those skilled in the art can refer to the content herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that those skilled in the art can modify or appropriately alter and combine the methods and applications herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0031] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0032] The present invention will be further described below in conjunction with the embodiments:

[0033] Preparation Example 1 Preparation of activated coagulation detection reagent

[0034] The components and concentrations of activated coagulation test reagent (pH = 7.0-8.5) are shown in Table 1

[0035] Table 1

[0036] Components Concentration (w / v) Tris 0.1％～0.6％ Kaolin 0.01％～0.05％ Gellan gum 0.1％～0.5％ <![CDATA[Mg 2+ ]]> 0.01％～2％ Gentamycin sulfate 0.01％～0.1％ Components Concentration (v / v) cephalin 0.1％～2％ water 98％～99.9％

[0037] Example 1

[0038] The activated coagulation test reagent is composed of 0.035% kaolin, 1% cephalin, 0.2% gellan gum, 0.15% Mg 2+ , 0.3% Tris, 0.05% gentamicin sulfate and the balance water.

[0039] Example 2

[0040] The activated coagulation test reagent is composed of 0.035% diatomaceous earth, 1% cephalin, 0.2% gellan gum, 0.15% Mg 2+ , 0.3% Tris, 0.05% gentamicin sulfate and the balance water.

[0041] Example 3

[0042] The activated coagulation test reagent is composed of 0.035% kaolin, 0.1% cephalin, 0.2% gellan gum, 0.15% Mg 2+ , 0.3% Tris, 0.05% gentamicin sulfate and the balance water.

[0043] Example 4

[0044] The activated coagulation test reagent is composed of 0.035% kaolin, 2% cephalin, 0.2% gellan gum, 0.15% Mg 2+ , 0.3% Tris, 0.05% gentamicin sulfate and the balance water.

[0045] Example 5

[0046] The activated coagulation test reagent is composed of 0.01% kaolin, 1% cephalin, 0.2% gellan gum, 0.15% Mg 2+ , 0.3% Tris, 0.05% gentamicin sulfate and the balance water.

[0047] Example 6

[0048] The activated coagulation test reagent is composed of 0.05% kaolin, 1% cephalin, 0.2% gellan gum, 0.15% Mg 2+ , 0.3% Tris, 0.05% gentamicin sulfate and the balance water.

[0049] Example 7

[0050] The activated coagulation test reagent is composed of 0.035% kaolin, 1% cephalin, 0.5% gellan gum, 0.15% Mg2+ , 0.3% Tris, 0.05% gentamicin sulfate and the balance water.

[0051] Example 8

[0052] The activated coagulation test reagent is composed of 0.035% kaolin, 1% cephalin, 0.1% gellan gum, 0.15% Mg 2+ , 0.3% Tris, 0.05% gentamicin sulfate and the balance water.

[0053] Comparative Example 1

[0054] The activated coagulation detection reagent comprises 0.035% kaolin, 1% cephalin, 0.3% Tris, 0.05% gentamicin sulfate and the balance water.

[0055] Comparative Example 2

[0056] A thrombelastogram activated coagulation detection kit (coagulation method) in the prior art.

[0057] Preparation of calcium chloride reagent: The calcium chloride reagent contains CaCl2 and gentamicin sulfate, the CaCl2 concentration is 0.1-1 mol / L, preferably 0.2 mol / L, and the gentamicin sulfate concentration is 0.01%-0.1%, preferably 0.05%.

[0058] Experimental Example 1 Feasibility Analysis

[0059] Blood samples from the same source were tested using the detection reagents of Examples 1-8 and Comparative Examples 1-2. The detection process included the following steps:

[0060] 1. On the corresponding thromboelastography software interface, enter the patient's name, test name, and select "CK-Citrated Kaolin" in the drop-down box for the test type.

[0061] 2. Load a set of ordinary cups on each channel of the thromboelastograph. Use a 1 mL pipette to transfer 1 mL of sodium citrate anticoagulated whole blood sample into the activated coagulation test reagent bottle. Invert the reagent bottle 5 times to mix thoroughly before use.

[0062] 3. Pipette 20 μL of calcium chloride reagent into the bottom of a standard cup.

[0063] 4. Pipette 340 μL of blood from the activated coagulation test reagent bottle into a regular cup.

[0064] 5. Move the cup to the test position and start the test.

[0065] 6. The coagulation process is completed in about 30 minutes. If the fibrinolysis process is to be observed, it needs to be extended to 1 hour.

[0066] 7. After the test is completed, click "Stop" on the software interface, remove the disposable ordinary cup, and discard it according to laboratory requirements.

[0067] The test results are shown in Table 2:

[0068] ① R value (clotting time) reflects the comprehensive effect of coagulation factors participating in the coagulation initiation process;

[0069] ②K value (clot formation time) is generally considered to represent the amplification stage of thrombin activation, which mainly leads to the solidification of blood clots through fibrinogen cleavage and fibrin polymerization;

[0070] ③Angle value (blood coagulation rate) is the result of the joint action of reactive fibrin and platelets when the blood clot begins to form. At this time, the function of fibrinogen is dominant. ④MA value (blood clot strength) reflects the maximum strength of the forming blood clot and the stability of blood clot formation, which is mainly affected by two factors: platelets and fibrin. Among them, the role of platelets is greater than that of fibrinogen, accounting for about 80%.

[0071] Table 2

[0072] experimental group R(min) K(min) Angle(deg) MA(mm) Example 1 6.8 1.9 65.1 60.1 Example 2 6.3 1.6 66.3 60.3 Example 3 7.2 2.1 63.1 59.6 Example 4 5.8 1.8 68.9 62.5 Example 5 8.7 2.6 56.4 57.1 Example 6 4.8 1.3 69.6 63.5 Example 7 7.5 2.3 62.5 58.8 Example 8 6.8 1.9 64.1 61.5 Comparative Example 1 6.8 1.9 65.3 60.5 Comparative Example 2 6.7 1.8 65.5 60.3

[0073] Results: The test results of Example 1 and Example 8 of the detection reagent of the present invention are similar to those of the commercially available detection reagent, indicating that the detection reagent of the present invention can be effectively applied to the thromboelastography instrument, but the stability of the reagent of Example 1 and the commercially available reagent cannot be explained, and subsequent stability exploration is carried out. The test results of Examples 2-7 are quite different from those of the commercially available detection reagent, indicating that Examples 2-7 are worse than Examples 1 and 8, and Examples 1 and 8 are subsequently selected for subsequent exploration.

[0074] Experimental Example 2 Stability Evaluation

[0075] The stability assessment process is as follows:

[0076] 1. The detection reagents in Example 1 and Example 8 and Comparative Examples 1-2 were placed at 37° C. for accelerated stabilization for 4 weeks.

[0077] 2. Take reagents from the same batch for quality control testing on days 0, 7, 14, and 28 of accelerated stabilization at 37°C.

[0078] The quality control of the same batch was tested using the detection reagents of Example 1 and Example 8 and Comparative Examples 1-2, respectively. The test results are shown in Table 3:

[0079] Table 3

[0080]

[0081]

[0082] Results: The detection reagent in Example 1 contains gellan gum as a stabilizer and is accelerated and stabilized at 37°C for 4 weeks. The deviation of the samples from the same batch is still very small. However, Example 8, which adds less gellan gum, and Comparative Example 1, which does not add gellan gum as a stabilizer, have larger deviations, indicating poor stability. The smaller deviation of the detection results of Example 1 than that of Comparative Example 2 indicates that the stability is better than that of commercially available detection reagents. Using gellan gum as a stabilizer can make the activated coagulation detection reagent more stable.

[0083] Experimental Example 3 Repeatability Evaluation

[0084] Repeatability evaluation process: The detection reagents in Example 1 and Comparative Examples 1-2 were subjected to quality control tests on the same batch ten times over 28 days.

[0085] The quality control of the same batch was tested using the detection reagents of Example 1 and Comparative Examples 1-2, respectively. The test results are shown in Table 4:

[0086] Table 4

[0087]

[0088]

[0089]

[0090] Results: Example 1 was accelerated at 37°C for 4 weeks, and the CV of the same batch of samples was very small when tested ten times. The CV of the test results of Example 1 was smaller than that of Comparative Examples 1-2, indicating that Example 1 has good repeatability and uniformity, is not easy to precipitate, and is superior to commercially available detection reagents.

[0091] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An activated coagulation detection reagent, characterized in that: It is composed of water, 0.3% (w / v) Tris, 0.035% (w / v) kaolin, 1% (v / v) phospholipids, 0.2% (w / v) gellan gum, 0.15% (w / v) magnesium chloride and 0.05% (w / v) gentamicin sulfate.

2. Use of the activated coagulation detection reagent according to claim 1 in preparing a coagulation method activated coagulation detection kit.

3. A coagulation activated coagulation detection kit, characterized in that: The activated coagulation detection reagent comprises the activated coagulation detection reagent according to claim 1 and calcium chloride, wherein the concentration of the calcium chloride is 0.1-1 mol / L.

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