An abnormal coagulation quality control product and its application

Abstract

The present invention relates to the field of biotechnology, and particularly to an abnormal coagulation quality control product and its application. The abnormal coagulation quality control product prepared by the present invention meets the target value requirements of APTT, PT, FIB, TT, and AT-III, and can be used for quality control during the detection of PT, APTT, FIB, TT, and AT-III reagent kits, for evaluating or verifying the analysis deviation of the detection system caused by changes in reagents or analytical instruments, thereby ensuring the precision of the detection results; at the same time, the coagulation quality control product has good reconstitution stability after lyophilization, can replace imported products for quality control of coagulation item detection, and is suitable for popularization and application in clinical practice.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an abnormal coagulation quality control product and its application. Background Art

[0002] Coagulation function tests mainly include prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT), and antithrombin III (AT-III). Coagulation function tests can reflect whether the intrinsic and extrinsic coagulation pathways are abnormal, whether there is hyperfibrinolysis and thrombosis. It is a necessary test item for screening the coagulation mechanism before surgery, early diagnosis of thrombotic diseases, and detection of thrombolytic therapy. The test results need to be fast and accurate. The commonly used routine coagulation tests play a very important role in clinical diagnosis, surgery, treatment, and prognosis. Therefore, it is extremely important to carry out coagulation quality control.

[0003] At present, the main coagulation quality control products on the market have few test items, only for two items of prothrombin time (PT) and activated partial thromboplastin time (APTT), or three items of prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB). Moreover, most of them are imported products from abroad, with poor stability, high prices, and are not easily available. Summary of the Invention

[0004] In view of this, the present invention provides an abnormal coagulation quality control product and its application. This abnormal coagulation quality control product has no matrix effect, high stability, and the values of five coagulation tests of PT, APTT, TT, FIB, and AT-III are within the abnormal coagulation index range. It is suitable for quality control during the detection process of the kit, and can evaluate or verify the analysis deviation of the detection system caused by changes in reagents or analytical instruments, thereby ensuring the precision of the test results.

[0005] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0006] A preparation method of an abnormal coagulation quality control product, comprising:

[0007] Taking mixed human plasma, adsorbing with an adsorbent, and centrifuging to obtain the first plasma;

[0008] Diluting the first plasma to obtain the second plasma;

[0009] Mixing the second plasma with the mixed human plasma that has not been adsorbed by the adsorbent in a ratio of 1:(1-2) to obtain a third plasma mixture system, and freeze-drying to obtain an abnormal coagulation quality control product; after testing, the values of PT, APTT, TT, FIB, and AT-III are all within the abnormal coagulation index range;

[0010] The buffer solution is a HEPES buffer solution containing NaCl, NaN3, sucrose, glycine, BSA, trehalose and CMC-Na.

[0011] In some embodiments, the mixing ratio of the second plasma to the mixed human plasma not adsorbed by the adsorbent is 1:1 or 1:2.

[0012] In some embodiments, in the mixing system, the concentrations of the components are as follows: 0.1 - 0.5 M NaCl, 1 - 5 g / L NaN3, 5 - 20 g / L sucrose, 5 - 20 g / L glycine, 2 - 8 g / L BSA, 5 - 20 g / L trehalose, 0.001 - 0.008 vol% CMC-Na, and 50 - 200 mM HEPES buffer solution with a pH of 7.4 - 8.0.

[0013] In some specific embodiments, in the mixing system, the concentrations of the components are as follows: 0.1 M NaCl, 1 g / L NaN3, 20 g / L sucrose, 5 g / L glycine, 5 g / L BSA, 10 g / L trehalose, 0.005 vol% CMC-Na, and 50 mM HEPES buffer solution with a pH of 7.4.

[0014] In some embodiments, the adsorbent is one or more of barium chloride, barium sulfate and aluminum hydroxide.

[0015] In some embodiments, the mass percentage concentration of the adsorbent is 20% - 30%.

[0016] In some specific embodiments, the mass percentage concentration of the adsorbent is 22%.

[0017] The present invention also provides an abnormal coagulation quality control product prepared by the preparation method of the present invention.

[0018] The present invention also provides the application of the coagulation quality control product in the preparation of coagulation detection reagents.

[0019] Among them, the indexes of the coagulation detection include at least one of PT, APTT, TT, FIB, and AT-III.

[0020] The present invention provides an abnormal coagulation quality control product and its application. The abnormal coagulation quality control product prepared by the present invention meets the target value requirements of APTT, PT, FIB, TT and AT-III, and can be used for quality control in the detection process of PT, APTT, FIB, TT and AT-III reagent kits, and is used to evaluate or verify the analysis deviation of the detection system caused by changes in reagents or analytical instruments, so as to ensure the precision of the detection results; at the same time, the coagulation quality control product has good reconstitution stability after lyophilization, can replace imported products for quality control of coagulation item detection, and is suitable for popularization and application in clinical practice. Detailed implementation manners

[0021] The present invention provides an abnormal coagulation quality control product and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes, or appropriate changes and combinations, to the methods and applications in this article without departing from the content, spirit and scope of the present invention, so as to implement and apply the technology of the present invention.

[0022] PT: Prothrombin time (PT) assay is used to screen for deficiencies in coagulation factors (I, II, V, VII, X) in the extrinsic and common coagulation pathways, and is also the preferred monitoring index for oral warfarin treatment.

[0023] APTT: Activated partial thromboplastin time (APTT) assay is used to screen for deficiencies in coagulation factors (I, II, V, VIII, IX, X, XI, XII) in the intrinsic and common coagulation pathways, and can also be used for the detection of coagulation factor inhibitors (such as lupus anticoagulant) and the monitoring of heparin anticoagulant therapy and coagulation factor replacement therapy in hemophilia patients.

[0024] TT: Thrombin time (TT) assay, and the TT assay result is used to reflect the index of decreased FIB.

[0025] FIB: Fibrinogen (FIB) content assay can be used for the screening of disseminated intravascular coagulation, primary fibrinolysis, severe hepatitis, and hypo (non)-fibrinogenemia.

[0026] AT-III: Assay of antithrombin III. AT-III (AntithrombinIII), also known as antithrombin (antithrombin, AT), is one of the most important anticoagulant substances in plasma physiological coagulation inhibitors.

[0027] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0028] The following further elaborates the present invention in conjunction with embodiments:

[0029] Example 1 Preparation of Abnormal Quality Control Product

[0030] (1) Take 1 L of mixed human plasma, add 220 g of adsorbent (aluminum hydroxide) for adsorption, and obtain the first plasma after low-temperature centrifugation.

[0031] (2) Mix the first plasma and buffer in a volume ratio of 1:1, and obtain the second plasma after dilution.

[0032] (3) Mix the second plasma with the human plasma before adsorption (i.e., the human plasma that has not been adsorbed by the adsorbent) in a volume ratio of 1:1 to obtain a third plasma mixture system; ensure that the detected PT, APTT, FIB, TT, and AT-III values are all within the range of abnormal coagulation indexes; the third plasma mixture system is freeze-dried to obtain an abnormal coagulation quality control product.

[0033] In the buffer solution described in step (2), the concentrations of each component are as follows:

[0034] Table 1

[0035] Component Dosage HEPES 11.9661g NaCl 5.855g <![CDATA[NaN3]]> 1g Sucrose 10g Glycine 5g BSA 5g Trehalose 10g CMC-Na 0.05 mL <![CDATA[H2O]]> Up to 1 L

[0036] Preparation of the abnormal quality control product in Example 2

[0037] The difference from Example 1 is that the first plasma and the buffer solution are diluted in a volume ratio of 1:1.5, and other conditions are the same.

[0038] Preparation of the quality control product in Comparative Example 1

[0039] The difference from Example 1 is that the first plasma is not diluted, and other conditions are the same.

[0040] Detect the five coagulation items of PT, APTT, FIB, TT, and AT-III in the quality control products of Examples 1 to 2 and Comparative Example 1, and the results are shown in Table 3.

[0041] Table 3

[0042]

[0043] The results show that in the preparation process of the quality control product in Comparative Example 1, the first plasma was not diluted, and the five indexes of PT, APTT, FIB, TT, and AT-III were within the normal range, which did not meet the requirements of the abnormal quality control product.

[0044] Preparation of the abnormal quality control product in Example 3

[0045] The difference from Example 1 is that the second plasma and the unadsorbed human plasma are mixed in a volume ratio of 1:2, and other conditions are the same.

[0046] Detect the five coagulation items of PT, APTT, FIB, TT, and AT-III in the quality control product of Example 3, and the results are shown in Table 4.

[0047] Table 4

[0048]

[0049] Preparation of the quality control product in Comparative Example 2

[0050] The difference from Example 1 is that in steps (2) and (4), the buffer solution does not include BSA and CMC-Na, and the specific components are shown in Table 5:

[0051] Table 5

[0052] Component Dosage HEPES 11.9661g NaCl 5.855g <![CDATA[NaN3]]> 1g Sucrose 10g Glycine 5g Trehalose 10g <![CDATA[H2O]]> 1L Total volume 1L

[0053] Test Example 1 Reconstitution Stability Test

[0054] The quality control products of Example 3 of the present invention, commercially available quality control products, and the quality control products of Comparative Example 2 were accurately reconstituted with distilled water at the labeled volume, stored at room temperature (20 - 28 °C), sampled once every 4 hours, and the PT, APTT, TT, FIB, and AT-III values were detected. The test results are shown in the following table.

[0055] Table 6

[0056]

[0057] Table 7

[0058]

[0059] Linear regression was performed on the test data, and a two-sided t-test was conducted on the significance difference between the slope b and 0. Looking up the table, it can be seen that α = 0.05, ν = 7, tα(ν) = 2.365, and the P values are all greater than 0.05, indicating that after reconstitution, the PT, APTT, TT, FIB, and AT-III test values of the coagulation quality control product prepared by the present invention remain basically unchanged after being stored at room temperature for 36 hours. However, the PT, APTT, TT, and FIB test values of the commercially available human-derived quality control plasma and the quality control product of Comparative Example 2 changed significantly after being opened and reconstituted at room temperature for 27 hours. This fully proves that the quality control product of the present invention is better and more stable than the commercially available quality control product and the quality control product of Comparative Example 2.

[0060] Test Example 2 Long-Term Stability Test

[0061] The quality control products of Example 3 of the present invention, commercially available quality control products, and the quality control products of Comparative Example 2 were stored at 2 - 8 °C. One bottle was taken out every 3 months and accurately reconstituted with distilled water at the labeled volume, and the PT, APTT, FIB, TT, and AT-III values were detected. The test results are shown in the following table.

[0062] Table 8

[0063]

[0064] Table 9

[0065]

[0066]

[0067] Linear regression was performed on the detection data, and a two-sided t-test was conducted on the significant difference between the slope b and 0. Looking up the table, it can be seen that α = 0.05, ν = 16, and tα(ν) = 2.12. The results show that the calculated P values of the detection results of PT, APTT, TT, FIB, and ATIII of the quality control product of the present invention are all greater than 0.05, indicating that the quality control product prepared by the present invention is basically stable when stored at 2-8°C for 36 months, has good long-term stability, and is conducive to commercial production.

[0068] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing an abnormal coagulation quality control product, characterized in that, Comprising: Step 1: Take mixed human plasma, adsorb it with an adsorbent, and centrifuge to obtain the first plasma; Step 2: Dilute the first plasma with a buffer solution to obtain the second plasma; Step 3: Mix the second plasma with the mixed human plasma that has not been adsorbed by the adsorbent in a ratio of 1:(1 - 2) to obtain a third plasma mixture system; After detecting that the PT, APTT, TT, FIB, and AT-III values are all within the range of abnormal coagulation indicators, the third plasma mixture system is freeze-dried to obtain an abnormal coagulation quality control product; In Step 2, the buffer solution is a HEPES buffer solution containing NaCl, NaN3, sucrose, glycine, BSA, trehalose, and CMC-Na; The volume ratio of the first plasma to the buffer solution is 1 - 1.5:

1.

2. The preparation method according to claim 1, wherein, In the mixture system, the concentrations of each component are: 0.1 - 0.5 M NaCl, 1 - 5 g / L NaN3, 5 - 20 g / L sucrose, 5 - 20 g / L glycine, 2 - 8 g / L BSA, 5 - 20 g / L trehalose, 0.001 - 0.008 vol% CMC-Na, and 50 - 200 mM HEPES buffer solution with a pH of 7.4 - 8.

0.

3. The preparation method according to claim 1, characterized in that, In the mixture system, the concentrations of each component are: 0.1 M NaCl, 1 g / L NaN3, 20 g / L sucrose, 5 g / L glycine, 5 g / L BSA, 10 g / L trehalose, 0.005 vol% CMC-Na, and 50 mM HEPES buffer solution with a pH of 7.

4.

4. The preparation method according to claim 1, characterized in that, The adsorbent is one or more of barium chloride, barium sulfate, and aluminum hydroxide.

5. The preparation method according to claim 1, characterized in that, The mass percentage concentration of the adsorbent is 20% - 30%.

6. The preparation method according to claim 5, characterized in that, The mass percentage concentration of the adsorbent is 22%.

7. An abnormal coagulation quality control product prepared by the preparation method according to any one of claims 1 - 6.

8. Use of the coagulation quality control product prepared by the preparation method according to any one of claims 1 - 6 or the coagulation quality control product according to claim 7 in the preparation of a coagulation detection reagent.

9. The application according to claim 8, wherein, The indicators for the coagulation detection include at least one of PT, APTT, TT, FIB, and AT-III.

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