Blood coagulation quality control kit and preparation method thereof

Abstract

The invention relates to the technical field of medicine detection, and provides a blood coagulation quality control kit and a preparation method thereof, a detection solution of 1L of the blood coagulation quality control kit comprises the following components in concentration: 110.17-185.26 g / L of nicotinamide, 80-150 mM / L of trihydroxymethyl aminomethane, 1.02-3.17 g / L of a hirudin solution, 15-30 g / L of a stabilizer, 0.5-2.8 ml / L of a preservative, 20-50 g / L of an excipient and the balance of a solvent, and the solvent is normal human plasma. Compared with the prior art, the method has the advantages that the stability is improved, the application range is wide, the method can be suitable for coagulation tests of five items including PT, APTT, TT, FIB and AT III, the values of the coagulation tests of the five items including PT, APTT, TT, FIB and AT III are within coagulation indexes of normal people, and the quality control requirements of the coagulation tests of the five items including PT, APTT, TT, FIB and AT III are met.

Description

Technical Field

[0001] The present application belongs to the field of medical testing technology, and in particular, relates to a coagulation quality control kit and a preparation method thereof. Background Art

[0002] Coagulation function tests mainly include plasma prothrombin time (PT), fibrinogen (FIB), activated partial thromboplastin time (APTT) and plasma thromboplastin time (TT). Coagulation function tests can reflect whether the intrinsic and exogenous coagulation pathways are abnormal, whether there is hyperfibrinolysis and thrombosis. They are essential monitoring items for pre-operative screening of coagulation mechanisms, early diagnosis of thrombotic diseases, and monitoring of thrombolytic therapy.

[0003] When the PT, APTT, FIB, anticoagulant drug concentration monitoring, and antithrombin activity tests mentioned above are performed clinically, corresponding quality control products are required to ensure the reliability of the test. The matrix of these quality control products is plasma. Among the chemical components of plasma, water accounts for 90% to 92%, and the solute is mainly plasma protein, which is the main solid component in plasma, with a content of 60 to 80 g / L. In order to prevent plasma from being affected by certain physical and chemical factors, some foreign substances are often added to maintain the stability of plasma quality.

[0004] Currently, there are many types of quality control products, but their quality varies greatly. Therefore, there is an urgent need to develop a quality control product with stable quality. Summary of the invention

[0005] The purpose of the present application is to provide a coagulation quality control product kit and a preparation method thereof, aiming to solve the above-mentioned problems existing in existing coagulation quality control products.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In the first aspect, a coagulation quality control kit is provided, wherein the detection solution of 1L of the coagulation quality control kit comprises components of the following concentrations: 110.17-185.26 g / L nicotinamide, 9.69-18.17 g / L tris(hydroxymethyl)aminomethane, 1.02-3.17 g / L hirudin solution, 15-30 g / L stabilizer, 0.5-2.8 ml / L preservative, 20-50 g / L excipient, and the remainder is solvent, which is normal human plasma.

[0008] As a possible design, the stabilizer includes glycine.

[0009] As a possible design, the excipient includes sucrose and / or mannitol.

[0010] As one possible design, the preservative includes ProClin-300.

[0011] As a possible design, the detection solution of a 1L coagulation quality control kit includes components of the following concentrations: 152.7g / L nicotinamide, 11.15g / L tris(hydroxymethyl)aminomethane, 2.35g / L hirudin solution, 20g / L stabilizer, 1.0ml / L preservative, 40g / L excipient, and the remainder is solvent.

[0012] As a possible design, the excipient includes 60-80% by mass of sucrose and 20-40% by mass of mannitol.

[0013] As a possible design, the excipient includes 75% by mass of sucrose and 25% by mass of mannitol.

[0014] As a possible design, the detection solution of a 1L coagulation quality control kit includes components of the following concentrations: 152.7g / L nicotinamide, 11.15g / L tris(hydroxymethyl)aminomethane, 2.35g / L hirudin solution, 20g / L glycine, 30g / L sucrose, 10g / L mannitol, 1ml ProClin-300, and the remainder is normal human plasma.

[0015] In a second aspect, the present application provides a method for preparing a coagulation quality control kit, comprising adding nicotinamide, tris(hydroxymethylaminomethane), hirudin solution, a stabilizer and a preservative to plasma, dissolving and mixing them in sequence, and then freeze-drying them to obtain a freeze-dried powder of the coagulation quality control kit.

[0016] As a possible design, the normal human plasma is obtained by mixing collected blood and sodium citrate solution and then separating by centrifugation. The volume ratio of blood to sodium citrate solution is 9:1, and the concentration of sodium citrate solution is 3.2wt%.

[0017] The beneficial effects of the present invention are:

[0018] By adding nicotinamide, tris(hydroxymethylaminomethane), hirudin solution stabilizer, preservatives and excipients at the same time, not only the stability can be improved, but also the scope of application is wide, which can meet the normal quality control requirements of the five coagulation tests of PT, APTT, TT, FIB and AT III. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 is a linear regression graph of the activated partial thromboplastin time detection (or determination) kit in the embodiment of the present application;

[0021] Figure 2 It is a linear regression graph of the prothrombin time detection (or determination) PT kit in the embodiment of the present application;

[0022] Figure 3 is a linear regression graph of the thrombin time kit in the embodiment of the present application;

[0023] Figure 4 It is a linear regression diagram of the fibrinogen assay kit in the examples of the present application. DETAILED DESCRIPTION

[0024] The main component of existing coagulation quality control products is plasma. Among the chemical components of plasma, water accounts for 90% to 92%, and the solute is mainly plasma protein, which is the main solid component in plasma, with a content of 60 to 80 g / L. In order to extend the shelf life of plasma, some additives are often added to plasma. At present, there are many kinds of additives, and the quality of the quality control products obtained is also uneven. Therefore, it is urgent to develop a quality control product with stable quality.

[0025] The following describes the invention in conjunction with specific embodiments.

[0026] Example 1

[0027] The present embodiment discloses a coagulation quality control kit. The detection solution of 1L of the coagulation quality control kit includes components with the following concentrations: 152.7g / L nicotinamide, 11.15g / L hydroxymethylaminomethane, 2.35g / L hirudin solution, 2g / L stabilizer, 1.0ml / L preservative, 40g / L excipient, and the rest is normal human plasma.

[0028] The matrix effect analysis of the coagulation quality control kit prepared in Example 1 was performed as follows:

[0029] 1. Sample processing

[0030] For each project, 35 clinical samples were collected, including normal and abnormal value samples. The samples showed no hemolysis, jaundice and other phenomena and were frozen at -25℃ to -15℃ for analysis of the comparison system and the system to be tested.

[0031] 2. Reagent Information

[0032]

[0033] 3. Data Analysis

[0034] 3.1 Valid Data

[0035] The mean of three repeated measurements is taken as one sample (clinical sample or processed sample). The valid data must not be less than 20 groups of samples, otherwise it needs to be supplemented.

[0036] Outlier determination:

[0037] Any sample is judged as an outlier if its absolute difference or relative difference between the two system tests exceeds 4 times the mean of the absolute difference or relative difference between the two system tests of all samples.

[0038] 3.2 Regression analysis

[0039] With the comparison system measurement value as the X-axis and the test system measurement value as the Y-axis, a scatter plot is made for the effective data of the clinical sample measurement. If the effective data shows an obvious linear relationship and all data points are evenly distributed on both sides of the regression curve, the results are subjected to linear regression analysis.

[0040] 3.3 Calculate 95% confidence interval

[0041] According to formula (1), the two-sided 95% prediction interval of the Y value (valid data measured by the test system) of the clinical sample for a given X value (valid data measured by the comparison system) is calculated.

[0042] 3.4 Evaluation of matrix effects

[0043] The comparison system measurement value of the effective clinical sample is used as the X-axis, and the measurement value of the system to be tested is used as the Y-axis, and the scatter plot of the clinical sample and the regression curve and 95% confidence interval of the clinical sample measurement are drawn respectively.

[0044] The judgment standard is whether the measured value Y of the prepared sample on the system to be tested is included in the corresponding 95% confidence interval. If the Y value exceeds the upper limit of the prediction interval, it means that the prepared sample shows a positive matrix effect on the conventional measurement system relative to the standard substance; if the Y value is lower than the lower limit of the prediction interval, it means that the prepared sample shows a negative matrix effect on the conventional measurement system; if the Y value is included in the prediction interval, there is no matrix effect on the conventional measurement system. When all valid prepared samples have no matrix effect, the coagulation quality control product has no matrix effect. Conversely, when the number of valid prepared samples with matrix effect is greater than or equal to 1, the coagulation quality control product has a matrix effect.

[0045] 4. Activated partial thromboplastin time test (or determination) reagent (APTT for short)

[0046] The experimental data are shown in Table 1.

[0047] Table 1

[0048]

[0049]

[0050] Table 2 Outlier Check

[0051]

[0052]

[0053] Table 3

[0054] / E1 E1' E2 E2' E3 E3' E E' / 1.17 0.03 1.22 0.04 1.25 0.03 1.15 0.03 Acceptable limit 4.68 0.14 4.88 0.15 5.02 0.12 4.61 0.14

[0055] According to the data results, there are no outliers and all the data are valid.

[0056] Regression analysis

[0057] Calculate the mean of each method for clinical samples and samples prepared by quality control products, and draw a scatter plot with the effective measurement value of clinical samples on the comparison system as the x-axis and the effective measurement value of clinical samples on the system to be tested as the y-axis. The data points of clinical samples in the scatter plot show an obvious linear relationship, and all data points are evenly distributed on both sides of the regression curve. Perform linear regression analysis on the data, and the results are as follows: Figure 1 As shown in the figure, "○" represents a sample and "+" represents a prepared sample.

[0058] Calculate 95% confidence interval

[0059] Table 4 Calculation data of effective clinical samples

[0060]

[0061]

[0062] Table 5 Calculation data of effective preparation samples

[0063]

[0064] 5. Prothrombin time detection (or determination) PT kit

[0065] Table 6 Experimental data

[0066]

[0067]

[0068] Table 7 Outlier Check

[0069]

[0070]

[0071] Table 8

[0072] / E1 E1' E2 E2' E3 E3' E E' / 1.17 0.07 1.05 0.06 1.04 0.03 1.01 0.06 Acceptable limit 4.69 0.30 4.18 0.25 4.17 0.12 4.05 0.24

[0073] According to the data results, sample 16 is an outlier, and the data becomes valid data after removing the outlier.

[0074] Regression analysis

[0075] Calculate the mean of each method for clinical samples and samples prepared by quality control products, and draw a scatter plot with the effective measurement value of clinical samples on the comparison system as the x-axis and the effective measurement value of clinical samples on the system to be tested as the y-axis. The data points of clinical samples in the scatter plot show an obvious linear relationship, and all data points are evenly distributed on both sides of the regression curve. Perform linear regression analysis on the data, and the results are as follows: Figure 2 As shown, in the figure, "○" represents a sample and "+" represents a prepared sample.

[0076] Calculate 95% confidence interval

[0077] Table 9 Calculation data of effective clinical samples

[0078]

[0079]

[0080] Table 10 Calculation data of effective preparation samples

[0081]

[0082] 6. Thrombin time kit

[0083] Table 11 Experimental data

[0084]

[0085]

[0086] Table 12 Outlier Check

[0087]

[0088]

[0089] Table 13

[0090] / E1 E1' E2 E2' E3 E3' E E' / 0.95 0.04 0.98 0.04 1.20 0.03 1.02 0.04 Acceptable limit 3.79 0.15 3.94 0.16 4.79 0.12 4.07 0.16

[0091] According to the data results, samples 12 and 35 are outliers. After removing the outliers, the data are valid data.

[0092] Regression analysis

[0093] Calculate the mean of each method for clinical samples and samples prepared by quality control products, and draw a scatter plot with the effective measurement value of clinical samples on the comparison system as the x-axis and the effective measurement value of clinical samples on the system to be tested as the y-axis. The data points of clinical samples in the scatter plot show an obvious linear relationship, and all data points are evenly distributed on both sides of the regression curve. Perform linear regression analysis on the data, and the results are as follows: Figure 3 As shown in the figure, "○" represents a sample and "+" represents a prepared sample.

[0094] Calculate 95% confidence interval

[0095] Table 14 Calculation data of effective clinical samples

[0096]

[0097]

[0098] Table 15 Calculation data of effective preparation samples

[0099]

[0100] 7. Fibrinogen Assay Kit (FIB for short)

[0101] Table 16 Experimental data

[0102]

[0103]

[0104] Table 17 Outlier Check

[0105]

[0106]

[0107] Table 18

[0108] / E1 E1' E2 E2' E3 E3' E E' / 0.23 0.06 0.23 0.06 0.25 0.03 0.22 0.06 Acceptable limit 0.91 0.24 0.91 0.24 1.00 0.12 0.90 0.24

[0109] According to the data results, there are no outliers and all the data are valid.

[0110] Regression analysis

[0111] Calculate the mean of each method for clinical samples and samples prepared by quality control products, and draw a scatter plot with the effective measurement value of clinical samples on the comparison system as the x-axis and the effective measurement value of clinical samples on the system to be tested as the y-axis. The data points of clinical samples in the scatter plot show an obvious linear relationship, and all data points are evenly distributed on both sides of the regression curve. Perform linear regression analysis on the data, and the results are as follows: Figure 4 As shown in the figure, "○" represents a sample and "+" represents a prepared sample.

[0112] Calculate 95% confidence interval

[0113] Table 19 Calculation data of effective clinical samples

[0114]

[0115]

[0116] Table 20 Calculation data of effective preparation samples

[0117]

[0118] Matrix effect evaluation

[0119] It can be seen from the test results that the measured values ​​Y of the prepared samples on the system to be tested are all included in the corresponding 95% confidence interval. Therefore, there is no matrix effect in the coagulation quality control product.

[0120] Stability assessment

[0121] 1. Study on shelf life stability

[0122] The quality control kit prepared in Example 1 was sealed and stored at 2°C to 8°C, tested at time 0 and 6M, and operated strictly in accordance with the product instructions, and compared with the 0d test results (control). At the same time, 2 bottles were reconstituted, each bottle was tested 3 times, and stability studies were performed according to the requirements of Table 21.

[0123] Table 21 Shelf life stability plan

[0124]

[0125]

[0126] Table 22

[0127]

[0128]

[0129] It can be concluded from the above table that the quality control performance of the quality control kit remains stable when stored for 25 months.

[0130] 2. Stability study of quality control products after reconstitution

[0131] 2.1 Stability test of the quality control product after reconstitution in a sealed storage at 2℃~8℃.

[0132] 1) Evaluation requirements

[0133] After reconstitution, the quality control product is sealed and stored at 2℃~8℃, which is stable for 8h. Take the newly reconstituted quality control product and test it at 0h, 4h, 6h, 8h and 9h. The relative deviation of the mean value of the test results of the newly reconstituted quality control product is within ±10%.

[0134] 2) Experimental plan

[0135] The reconstituted quality control product was stored in a sealed container at 2°C ~ 8°C, and then two bottles were taken out for testing at 4h, 6h, 8h, and 9h, respectively. Each bottle was tested 3 times, and the mean was calculated and compared with the test result at 0h to calculate the relative deviation.

[0136] Table 23

[0137]

[0138] Standardize the values ​​of test controls.

[0139] Table 24

[0140]

[0141]

[0142] The results showed that after the quality control product was opened and reconstituted, it was stored in a sealed container at 2℃~8℃. The result was qualified after 9 hours, and the result did not change significantly after 9 hours. The stable time of the quality control product after opening the bottle and reconstituted was determined to be 8 hours.

[0143] 2.2 Stability test of the quality control product after reconstitution in a sealed storage at -25℃~-15℃.

[0144] Assessment Requirements

[0145] After reconstitution, the quality control product is sealed and stored at -25℃~-15℃, and it is stable for 7 days. Take the newly reconstituted quality control product for testing at time 0, 3d, 5d, 7d, and 8d. The relative deviation of the mean value of the test results of the newly reconstituted quality control product is within ±10%.

[0146] Trial plan

[0147] After reconstitution, the quality control products were sealed and stored at -25℃~-15℃. The reconstituted quality control products were taken for testing at 0, 3d, 5d, 7d and 8d. Two bottles were taken each time and the test was repeated 3 times for each bottle. The relative deviation of the mean value of the test results of the reconstituted quality control products at each time interval was calculated.

[0148] Table 25

[0149]

[0150] Standardize the values ​​of test controls.

[0151] Table 26

[0152]

[0153]

[0154] After the quality control product is opened and reconstituted, it is sealed and stored at -25℃ ~ -15℃. It is tested for 8 days according to the time intervals in the table. The relative deviation of the test results from the test results on day 0 is ≤10%, and the test results on the 8th day did not change significantly. Therefore, the quality control product is sealed and stored at -25℃ ~ -15℃ after opening the bottle and reconstituted. It is determined to be stable within 7d (days), which meets the experimental requirements.

[0155] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0156] In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present specification, the terms "including", "comprising", etc. refer to "including but not limited to". In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of the associated objects, indicating that there may be three relationships, for example, A and / or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e. a and b), ac, bc or abc, where a, b, c can be single or plural, respectively.

[0157] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A coagulation quality control kit, characterized in that: The detection solution of the 1L coagulation quality control kit includes components of the following concentrations: 110.17-185.26g / L nicotinamide, 9.69-18.17g / L tris(hydroxymethyl)aminomethane, 1.02-3.17g / L hirudin solution, 15-30g / L stabilizer, 0.5-2.8ml / L preservative, 20-50g / L excipient, and the remainder is solvent, which is normal human plasma.

2. The blood coagulation quality control kit according to claim 1, characterized in that: The stabilizer includes glycine.

3. The blood coagulation quality control kit according to claim 1, characterized in that: The excipients include sucrose and / or mannitol.

4. The coagulation quality control kit according to claim 1, characterized in that: The preservatives include ProClin-300.

5. The blood coagulation quality control kit according to any one of claims 1 to 4, characterized in that: The detection solution of 1L coagulation quality control kit includes the following components: 152.7g / L nicotinamide, 11.15g / L tris(hydroxymethyl)aminomethane, 2.35g / L hirudin solution, 20g / L stabilizer, 1.0ml / L preservative, 40g / L excipient, and the rest is solvent.

6. The blood coagulation quality control kit according to claim 5, characterized in that: The excipient contains 60-80% of sucrose and 20-40% of mannitol in mass percentage.

7. The blood coagulation quality control kit according to claim 6, characterized in that: The excipient contains 75% sucrose and 25% mannitol in mass percentage.

8. The blood coagulation quality control kit according to claim 1, characterized in that: The test solution of 1L coagulation quality control kit includes the following components: 152.7g / L nicotinamide, 11.15g / L tris(hydroxymethyl)aminomethane, 2.35g / L hirudin solution, 20g / L glycine, 30g / L sucrose, 10g / L mannitol, 1ml ProClin-300, and the remainder is normal human plasma.

9. A method for preparing the coagulation quality control kit according to any one of claims 1 to 8, characterized in that: Nicotinamide, tris(hydroxymethylaminomethane), hirudin solution, stabilizer and preservative are added to plasma, dissolved and mixed in sequence, and then freeze-dried to obtain freeze-dried powder of the coagulation quality control kit.

10. The method for preparing the coagulation quality control kit according to claim 9, characterized in that: The normal human plasma is obtained by mixing collected blood and sodium citrate solution and then centrifuging. The volume ratio of blood to sodium citrate solution is 9:1, and the concentration of sodium citrate solution is 3.2wt%.

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