Thrombin time quality control product and its preparation method and application

Abstract

The present invention relates to the field of biological preparations, and in particular to a thrombin time quality control product and a preparation method and application thereof. The present invention provides a thrombin time quality control product, the raw materials of which include blood, an anticoagulant and a freeze-dried protective solution, the prepared quality control product has good stability after reconstitution, the freeze-dried product has good long-term stability, and the freeze-dried product has stability of up to 33 months. In addition, the quality control material preparation process of the present invention is simple and low-cost, the raw materials are easily available, and it is suitable for mass production, and has good industrialization and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological preparations, and in particular to a thrombin time quality control product and a preparation method and application thereof. Background Art

[0002] Coagulation: short for blood coagulation, is the process of blood changing from a liquid state to a gel state, and is an important part of the hemostasis function of mammals. Coagulation is a process in which a series of coagulation factors are successively activated by enzymatic hydrolysis, and finally thrombin is generated to form a fibrin clot. The coagulation process often includes three pathways, the intrinsic coagulation pathway, the exogenous coagulation pathway and the common coagulation pathway.

[0003] Thrombin time: refers to the time it takes for blood to coagulate after standardized thrombin is added to plasma. In the common coagulation pathway, the generated thrombin converts fibrinogen into fibrin, which can be reflected by the thrombin time.

[0004] Coagulation factors are a series of proteins in mammalian blood that participate in the blood coagulation cascade reaction. They have homology among different mammals, and the blood coagulation process in animals is similar to that in humans.

[0005] The thrombin time quality control products currently used in China are all imported products. They are expensive, the raw materials are difficult to obtain, they are not suitable for mass production, and they are difficult to dissolve. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a thrombin time quality control product and a preparation method and application thereof, wherein the quality control product has low cost and good re-dissolution stability.

[0007] The invention provides a thrombin time quality control product, the raw materials of which include blood, an anticoagulant and a freeze-dried protective solution, wherein the freeze-dried protective solution includes a B-400 aqueous defoamer, nicotinamide, HEPES, sucrose, sodium laurate, sodium perfluorooctanoate, thimerosal, bovine serum albumin, glycine and NaOH; the B-400 aqueous defoamer, nicotinamide, HEPES, sucrose, sodium laurate, sodium perfluorooctanoate, thimerosal, bovine serum albumin, glycine and NaOH in the freeze-dried protective solution. The mass ratio of sodium thiomersal, bovine serum albumin and glycine is (5-10): (45.6-91.59): (123.6-357.45): (100-200): (0.555-0.111): (0.104-0.218): (2.5-5.0): (100-200): (25-50), and the pH is adjusted to 7.3-7.6 with NaOH.

[0008] In some embodiments, in the thrombin time quality control product described in the present invention, the concentration of each component in the lyophilized protective solution is B-400 aqueous defoaming agent of 5g / L~10g / L, nicotinamide of 45.6g / L~91.59g / L, HEPES of 123.6g / L~357.45g / L, sucrose of 100g / L~200g / L, sodium laurate of 0.555g / L~0.111g / L, sodium perfluorooctanoate of 0.104g / L~0.2180g / L, thimerosal of 2.5g / L~5.0g / L, bovine serum albumin of 100g / L~200g / L, glycine of 25g / L~50g / L and NaOH.

[0009] In other embodiments, in the thrombin time quality control product described in the present invention, the concentration of each component in the lyophilized protective solution is 10 g / L B-400 aqueous defoamer, 91.59 g / L nicotinamide, 357.45 g / L HEPES, 200 g / L sucrose, 0.111 g / L sodium laurate, 0.2180 g / L sodium perfluorooctanoate, 5.0 g / L thimerosal, 200 g / L bovine serum albumin, 50 g / L glycine and NaOH.

[0010] In the thrombin time quality control product of the present invention, the blood is pig blood.

[0011] In the thrombin time quality control product of the present invention, the anticoagulant is a 2wt% to 4wt% trisodium citrate aqueous solution.

[0012] In some embodiments, in the thrombin time quality control product described in the present invention, the anticoagulant is a 3.8 wt % trisodium citrate aqueous solution.

[0013] In the thrombin time quality control product of the present invention, the volume ratio of the blood containing the anticoagulant to the freeze-dried protective solution is (0.6-0.7): (0.3-0.4).

[0014] The pH value of the thrombin time quality control product of the present invention is 7.3 to 7.6. In some embodiments, the pH value of the thrombin time quality control product is 7.40.

[0015] The thrombin time quality control product provided by the present invention, its raw materials include blood, anticoagulant and freeze-dried protective solution, the quality control product not only has good stability after reconstitution, but also has good long-term stability of the freeze-dried product. Experiments show that the stability of its freeze-dried product can reach 33 months. It was found in the experiment that during the preparation of the quality control product, the selection of anticoagulant and freeze-dried protective solution had a significant impact on the quality of the quality control product. The applicant optimizes the selection and dosage of each component so that each component cooperates with each other and supports each other, and under the interaction, good stability is achieved together. The present invention also provides a preparation method of the thrombin time quality control product, which includes mixing the freeze-dried protective solution with blood containing an anticoagulant, and freeze-drying to obtain a thrombin time quality control product.

[0016] In the method of the present invention, the method for preparing the blood containing the anticoagulant includes: mixing the blood with the anticoagulant and then centrifuging to obtain plasma; the volume ratio of the blood to the anticoagulant is 9:1.

[0017] In the method described in the present invention, the centrifugal conditions include 2000g to 2500g and the centrifugal time is 20min to 40min.

[0018] In some embodiments, in the method described in the present invention, the centrifugation conditions include 2000g and centrifugation time of 30min.

[0019] The present invention provides a thrombin time quality control product, the raw materials of which include blood, an anticoagulant and a freeze-dried protective solution, the prepared quality control product has good stability after reconstitution, the freeze-dried product has good long-term stability, and the freeze-dried product has stability of up to 33 months. In addition, the quality control material preparation process of the present invention is simple and low in price, the raw materials are easily available, and it is suitable for mass production, and has good industrialization and clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation or the prior art description are briefly introduced below. Obviously, the drawings in the following description are some implementations of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work:

[0021] Figure 1 The results of the stability test of the thrombin time quality control product of the present invention and the commercially available thrombin time quality control product after opening a bottle are shown;

[0022] Figure 2 The long-term stability test results of the thrombin time quality control product of the present invention and the commercially available product are shown. DETAILED DESCRIPTION

[0023] The present invention provides thrombin time quality control product and preparation method and application thereof, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention, to realize and apply the technology of the present invention.

[0024] The purpose of the present invention is to provide a method for preparing a thrombin time quality control product using porcine plasma. To achieve the above purpose, the present invention adopts the following technical scheme:

[0025] Plasma was placed in a plastic beaker, freeze-dried protective solution was added to dilute the plasma, and then freeze-dried to obtain a long-term stable thrombin time quality control product.

[0026] In the embodiment of the present invention, the plasma is porcine plasma, and the preparation method thereof comprises: collecting blood from the jugular vein of a porcine, adding an anticoagulant to the blood at a ratio of 9:1, wherein the anticoagulant is a 2% to 5% trisodium citrate aqueous solution, centrifuging the blood with the anticoagulant for 30 minutes at a relative centrifugal force of 2000 g to 2500 g, and carefully separating and absorbing the upper plasma.

[0027] The formula of the freeze-dried protective solution is that the volume ratio of the freeze-dried protective solution to the plasma is (0.3-0.4): (0.6-0.7).

[0028] Serial number Name of raw materials concentration 1 B-400 water-based defoamer 5~10g / L 2 Niacinamide 45.6~91.59g / L 3 HEPES 123.6~357.45g / L 4 sucrose 100~200g / L 5 Sodium laurate 0.555~0.111g / L 6 Sodium perfluorooctanoate 0.104~0.2180g / L 7 Thimerosal 2.5~5.0g / L 8 BSA 100~200g / L 9 Glycine 25~50g / L 10 10M NaOH 60~120mL

[0029] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0030] Example 1

[0031] 1. Blood collected from the jugular vein of a pig was added with an anticoagulant (3.8 wt% sodium citrate trihydrate solution) at a ratio of 9:1. The blood with the anticoagulant was centrifuged for 30 minutes at a relative centrifugal force of 2000 g, and the upper plasma was carefully separated and absorbed.

[0032] 2. Take a certain amount of upper pig plasma and place it in a plastic beaker, add protective solution to dilute the pig plasma to 30% (volume fraction), and obtain quality control products by freeze-drying.

[0033] The formula of the freeze-dried protective solution is as follows:

[0034]

[0035]

[0036] Comparative Example 1

[0037]

[0038] Effect verification

[0039] Using Example 1, Comparative Example 1 and commercially available quality control products as samples, the stability of the thrombin time quality control product was tested.

[0040] 1. Open bottle stability test: The thrombin time quality control product prepared in Example 1 was accurately re-dissolved and stored at room temperature (20°C to 28°C). Samples were taken every 24 hours to test the thrombin time quality control product. The test results are as follows: Figure 1 , Table 1. The results show that the thrombin time quality control product prepared by the present invention is relatively stable under room temperature storage conditions after reconstitution, and the commercial quality control product has a decreased concentration from the seventh day after opening the bottle for storage at room temperature, indicating that the thrombin time quality control product prepared by the present invention has better stability.

[0041] Table 1: Opening stability test results

[0042] Storage time (days) Example 1 Quality Control Product(s) Commercially available quality control products(s) Comparative Example 1 Quality Control Product(s) 0 45.2 20.1 45.9 1 44.7 22.1 43.5 2 45.1 21.0 44.0 3 46.2 23.0 45.7 4 44.0 21.0 46.2 5 45.2 21.1 47.1 6 45.8 20.1 47.7 7 46.2 21.0 47.3 8 47.8 23.1 48.7 9 46.5 24.1 49.1 10 45.5 24.6 48.9

[0043] The regression equation of the quality control product of the present invention is: y=0.169x+44.81, and a bilateral t test is performed on the significant difference between the slope b and 0. According to statistical calculation, t b =1.985, P>0.05, indicating that the thrombin time quality control product prepared by the present invention is basically stable after being reconstituted and stored at room temperature for 10 days. The regression equation of the commercial quality control product is: y=0.290x+20.48t b =2.347, P < 0.05, indicating that the thrombin time test value of the commercially available quality control product after being opened and stored at room temperature for 10 days changed significantly, and the regression equation of the control quality control product was y = 0.510x + 44.18t b =6.77, P < 0.05, indicating that the stability of the quality control product after removing some protective agents is poor, and Figure 1 It can be seen that the thrombin time test value was significantly prolonged starting from the 7th day.

[0044] It can be seen that the thrombin time quality control product prepared by the present invention has better opening stability than commercially available products. For hospital clinical laboratories, especially grassroots hospitals with small business volume, in the quality control work of thrombin time detection, the quality control product can be stored for a longer time after opening the bottle, which can reduce reagent waste, reduce detection costs, and bring convenience to work.

[0045] 2. Long-term stability test: The lyophilized product of the thrombin time quality control product prepared in Example 1 was stored at 2-8°C, and one bottle was taken out every 3 months for accurate reconstitution, and the concentration of the thrombin time quality control product was tested. Figure 2 , Table 2. The results show that the lyophilized product of the thrombin time quality control product prepared by the present invention is still relatively stable after 33 months, which is conducive to commercial production.

[0046] Table 2: Long-term qualitative test results

[0047] Storage time (days) Example 1 Quality Control Product(s) Commercially available quality control products(s) Comparative Example 1 Quality Control Product(s) 0 45.8 21.1 45.6 3 45.6 22.2 46.7 6 44.2 21.3 46.2 9 46.0 21.0 45.9 15 45.2 20.1 47.7 18 44.8 22.0 47.1 21 44.7 20.1 48.1 24 46.5 21.2 48.3 27 45.2 19.8 48.9 30 46.9 18.8 49.7 33 45.4 18.2 50.1

[0048] Through linear regression analysis, y = 0.017x + 45.2, a bilateral t test was performed on the significant difference between the slope b and 0. According to statistical calculations, t b =0.728, P>0.05, indicating that the thrombin time quality control product prepared by the present invention is basically stable when stored at +2 to +8°C for 33 months, has good long-term stability, and is conducive to commercial production.

[0049] The regression equation of commercially available quality control products is: y = 0.101x + 20.357, t b =2.337, P < 0.05, indicating that the commercially available quality control products showed significant changes after being stored at +2 to +8℃ for 33 months, and Figure 2 It can be seen that starting from the 27th month, the thrombin time test value was significantly prolonged.

[0050] The regression equation for the control product is: y = 0.435x + 45.49t b =10.14, P < 0.05, indicating that the stability of the quality control product after removing some protective agents is poor.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A thrombin time quality control product, characterized in that: Its raw materials include blood, anticoagulant and freeze-dried protective solution. The anticoagulant is a 2wt% to 4wt% trisodium citrate aqueous solution; The freeze-drying protective solution comprises B-400 aqueous defoaming agent, nicotinamide, HEPES, sucrose, sodium laurate, sodium perfluorooctanoate, thimerosal, bovine serum albumin, glycine and NaOH; the mass ratio of B-400 aqueous defoaming agent, nicotinamide, HEPES, sucrose, sodium laurate, sodium perfluorooctanoate, thimerosal, bovine serum albumin and glycine in the freeze-drying protective solution is (5-10): (45.6-91.59): 123.6-357.45: 100-200: 0.555-0.111: 0.104-0.2180: 2.5-5.0: 100-200: 25-50, and the pH is adjusted to 7.3-7.6 with NaOH.

2. The thrombin time quality control product according to claim 1, characterized in that: The concentrations of the components in the freeze-dried protective solution are 5 g / L to 10 g / L of B-400 aqueous defoamer, 45.6 g / L to 91.59 g / L of nicotinamide, 123.6 g / L to 357.45 g / L of HEPES, 100 g / L to 200 g / L of sucrose, 0.555 g / L to 0.111 g / L of sodium laurate, 0.104 g / L to 0.2180 g / L of sodium perfluorooctanoate, 2.5 g / L to 5.0 g / L of thimerosal, 100 g / L to 200 g / L of bovine serum albumin, 25 g / L to 50 g / L of glycine and NaOH.

3. The thrombin time quality control product according to claim 1, characterized in that: The blood is pig blood.

4. The thrombin time quality control product according to claim 1, characterized in that: The anticoagulant is a 3.8 wt % trisodium citrate aqueous solution.

5. The thrombin time quality control product according to claim 1, characterized in that: The volume ratio of the blood containing the anticoagulant to the freeze-dried protective solution is (0.6-0.7): (0.3-0.4).

6. The method for preparing the thrombin time quality control product according to any one of claims 1 to 5, characterized in that: The method comprises mixing the freeze-dried protective solution with blood containing an anticoagulant, and then freeze-drying the mixture to obtain a thrombin time quality control product.

7. The method according to claim 6, characterized in that The method for preparing the blood containing the anticoagulant comprises: mixing the blood with the anticoagulant and then centrifuging to obtain plasma; the volume ratio of the blood to the anticoagulant is 9:

1.

8. The method according to claim 7, characterized in that The centrifugal conditions include 2000g to 2500g and a centrifugal time of 20min to 40min.

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