A clinical biochemical composite quality control product and a preparation method thereof

Abstract

The application discloses a clinical biochemical composite quality control product, which comprises detection item raw materials, a matrix liquid and a protective agent; the detection item raw materials are alkaline phosphatase, amylase, sodium glycocholate, cholinesterase, creatinine, leucine aminopeptidase, lactate dehydrogenase, lipase, triglyceride, cholesterol, uric acid, urea, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, glucose, alpha-hydroxybutyric acid dehydrogenase, sodium hydrogen phosphate, magnesium chloride, calcium chloride, ferrous chloride, bilirubin, pancreatic amylase and sodium bicarbonate; the matrix liquid is bovine serum albumin and human serum albumin; and the protective agent is dithiothreitol (DTT), mannitol, trehalose, sodium chloride, a composite enzyme stabilizer AES and sodium azide. The quality control product can realize the detection of 27 clinical biochemical items, and the freeze-drying process is adopted, so that the freeze-dried composite quality control product has low viscosity, is easy to be re-dissolved and mixed, has high stability and has small matrix effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of in vitro diagnostic reagents, and particularly relates to a clinical biochemical composite quality control product and a preparation method thereof. BACKGROUND

[0002] With biochemical analyzers and biochemical reagents being widely applied to clinical medical examination, a plurality of matched clinical biochemical quality control products are particularly important for quality control. A laboratory draws a quality control chart by measuring the quality control product to monitor the stability of the matched detection project in the laboratory detection system (factors such as instruments, reagents, staff and environment), and timely discovers the out-of-control state of the detection system. If there is an out-of-control state, corresponding measures are taken to correct it in time to ensure that the detection system is in a normal state. The quality of the quality control product directly relates to the diagnosis result of the in vitro diagnostic reagent.

[0003] At present, there are few composite quality control products on the market, and most of them are quality control products for a single detection project. However, the use of single quality control products brings certain inconvenience to operation and use, and the main problems are as follows: 1. Few composite types: due to high price and poor product stability, most quality control products on the market have few composite projects, which cannot provide great convenience for clinical use. 2. Poor stability and insufficient uniformity: most biochemical reactions in biochemical reagents are catalyzed by enzymes, and the stability of enzymes determines the stability of reagents. The stability of enzymes in liquid has always been a well-known problem, especially in biochemical reagents containing enzymes. It is a big problem to ensure that the activity of enzymes does not change with time. Especially in composite quality control products, the complex composition increases the risk of enzyme damage, resulting in poor stability and non-uniformity of the quality control product. 3. Matrix effect: factors such as the design of the instrument, the composition of the reagent, the principle of the test method, the composition and processing technology of the quality control material, etc. can produce matrix effect. If the matrix effect cannot be reduced or eliminated, it will affect the correctness of the measurement result. Therefore, it is particularly important to develop biochemical composite quality control products with good stability and uniformity, which can provide great convenience for clinical use. SUMMARY

[0004] To solve the above problems, the application provides a clinical biochemical composite quality control product and a preparation method thereof. The quality control product can realize the detection of 27 clinical biochemical projects, and the freeze-drying process is adopted. The freeze-dried composite quality control product has low viscosity, is easy to reconstitute and mix, has strong stability, and has small matrix effect.

[0005] To achieve the above purpose, the application provides the following scheme:

[0006] A clinical biochemical composite quality control product, which comprises detection project raw materials, a matrix liquid and a protective agent.

[0007] The detection item raw material is: alkaline phosphatase 30-400 U / L, amylase 20-400 U / L, sodium glycochenodeoxycholate 2-80 μmol / L, cholinesterase 2000-13000 U / L, creatinine 40-300 μmol / L, leucine aminopeptidase 10-100 U / L, lactate dehydrogenase 50-500 U / L, lipase 20-100 U / L, triglyceride 0.4-5 mmol / L, cholesterol 3.12-10.4 mmol / L, uric acid 100-600 μmol / L, urea 1.43-20 mmol / L, alanine aminotransferase 7-100 U / L, aspartate aminotransferase 12-100 U / L, gamma glutamyl transferase 10-100 U / L, glucose 2-10 mmol / L, alpha-hydroxybutyric acid dehydrogenase 30-300 U / L, sodium hydrogen phosphate 1-4 mg / dL, magnesium chloride 0.5-1.5 mg / dL, calcium chloride 2-3.75 mmol / L, ferrous chloride 6-50 μmol / L, bilirubin 2-100 μmol / L, pancreatic amylase 100-1000 U / L, sodium bicarbonate 10-40 mmol / L;

[0008] The matrix liquid is: bovine serum albumin 30-90 g / L, human serum albumin 30-90 g / L;

[0009] The protective agent is: dithiothreitol (DTT) 1-5 g / L, mannitol 10-50 g / L, trehalose 10-50 g / L, sodium chloride 1-10 g / L, complex enzyme stabilizer AES 5-10 g / L, sodium azide 0.5 g / L.

[0010] As a preferred technical solution, the composite quality control product comprises a detection item raw material, a matrix liquid and a protective agent;

[0011] The detection item raw material is: alkaline phosphatase 30-400 U / L, amylase 20-400 U / L, sodium glycochenodeoxycholate 2-80 μmol / L, cholinesterase 2000-13000 U / L, creatinine 40-300 μmol / L, leucine aminopeptidase 10-100 U / L, lactate dehydrogenase 50-500 U / L, lipase 20-100 U / L, triglyceride 0.4-5 mmol / L, cholesterol 3.12-10.4 mmol / L, uric acid 100-600 μmol / L, urea 1.43-20 mmol / L, alanine aminotransferase 7-100 U / L, aspartate aminotransferase 12-100 U / L, gamma glutamyl transferase 10-100 U / L, glucose 2-10 mmol / L, alpha-hydroxybutyric acid dehydrogenase 30-300 U / L, sodium hydrogen phosphate 1-4 mg / dL, magnesium chloride 0.5-1.5 mg / dL, calcium chloride 2-3.75 mmol / L, ferrous chloride 6-50 μmol / L, bilirubin 2-100 μmol / L, pancreatic amylase 100-1000 U / L, sodium bicarbonate 10-40 mmol / L;

[0012] The matrix liquid is: bovine serum albumin 30g / L, human serum albumin 50g / L;

[0013] The protective agent is: dithiothreitol (DTT) 3g / L, mannitol 50g / L, trehalose 50g / L, sodium chloride 9g / L, complex enzyme stabilizer AES 5g / L, sodium azide 0.5g / L.

[0014] The preparation method of the clinical biochemical composite quality control product of the application comprises the following steps:

[0015] (1) According to the raw material formula, each component is prepared with distilled water in advance for standby;

[0016] (2) Under the stirring state, bovine serum albumin and human serum albumin are added and stirred until completely dissolved;

[0017] (3) After the bovine serum albumin and human serum albumin are completely dissolved, a filter membrane is used to filter into another clean preparation container;

[0018] (4) The protective agent, dithiothreitol, mannose, trehalose, sodium chloride, complex enzyme stabilizer AES and sodium azide are sequentially added into the solution of step (3) and stirred until completely dissolved;

[0019] (5) After the substances of step (4) are completely dissolved, a basic stable solution of the composite quality control product is formed, which is filtered into another clean sealable storage container by a filter membrane and stored at 2-8℃ for standby;

[0020] (6) The detection item raw material is added into the solution of step (5) and stirred uniformly to form a multi-item detection composite quality control product;

[0021] (7) The multi-item detection composite quality control product obtained in step (6) is quantitatively packaged;

[0022] (8) The packaged quality control product is subjected to freeze-drying operation to obtain the finished product, and the specific operation is that it is pre-frozen for 4-5h at a temperature of-40℃, vacuumizing is performed after the freeze-drying pressure reaches 150ubar, and then drying is performed, and the drying process continues until the temperature of the freeze-drying machine reaches 25℃, so that the freeze-dried powder of the clinical biochemical composite quality control product is obtained.

[0023] As a preferred technical solution, the stirring of steps (2) and (4) is at a speed of 400-500r / min for 30-40min until completely dissolved.

[0024] The filter membrane used in the application has a pore size of 0.22μm.

[0025] The lipid quality control product can be placed at 2-8℃ for long-term storage.

[0026] The present application has the following beneficial effects:

[0027] 1、The composite quality control product produced by the present application can realize the detection of 27 clinical biochemical items, has wide adaptation range, and has the characteristics of good uniformity, strong anti-interference, high accuracy, good stability, low cost and the like.

[0028] 2、The clinical biochemical composite quality control product of the present application uses multiple composite stabilizers and composite protective agents, has the protection and stabilization effects on protein, multiple enzymes and other substances in the components, and the amount of the protective agent is less and the stability is stronger compared with other existing composite quality control products.

[0029] 3、The present application uses the freeze-drying technology, which is more beneficial to the long-term preservation of various biochemical components in the freeze-drying state; and the freeze-dried quality control product developed by the present application has low viscosity, is easy to be reconstituted and mixed, has strong stability, and has small matrix effect.

[0030] 4、The clinical biochemical composite quality control product of the present application adds human and animal serum combination in the components, so as to reduce the matrix effect in clinical use and ensure the test accuracy. DETAILED DESCRIPTION

[0031] The present application will be further described below in combination with examples:

[0032] Example 1

[0033] A clinical biochemical composite quality control product, comprising detection item raw materials, matrix liquid and protective agents, and the specific formula is as follows:

[0034]

[0035]

[0036] The specific preparation method of the clinical biochemical composite quality control product of the present application comprises the following steps:

[0037] Step 1: According to the preparation amount of the above formula, distilled water is added to a preparation container for standby.

[0038] Step 2: Under stirring, bovine serum albumin and human serum albumin are added, and after the addition of the materials, stirring is carried out at 400 r / min for 30 minutes until complete dissolution.

[0039] Step 3: After complete dissolution, the solution is filtered into another clean preparation container by using a filter membrane with a pore size of 0.22 μm.

[0040] Step 4: Dithiothreitol, mannose, trehalose, sodium chloride, composite enzyme stabilizer AES and sodium azide are sequentially added to the above solution, and stirring is carried out at 400 r / min for 30 minutes until complete dissolution.

[0041] Step 5, after complete dissolution, the complex quality control basic stable liquid is formed, and is filtered into another clean sealable storage container with a filter membrane with a pore size of 0.22 μm and is placed at 2-8℃ for standby;

[0042] Step 6, the remaining raw materials of the detection items are added to form a multi-item detection complex quality control;

[0043] Step 7, the clinical complex biochemical quality control is quantitatively packaged in 5.0 ml brown glass bottles;

[0044] Step 8, the packaged quality control is freeze-dried according to the following steps: pre-freeze-drying at -40℃ for 4-5 h, vacuumizing after the pressure reaches 150 uba (about 2 hours), drying (the drying process continues until the temperature of the freeze-drying machine reaches 25℃, which takes more than 12 hours, usually 24 hours), and completion.

[0045] When the quality control is used for inspection, the quality control must be reconstituted according to the following steps:

[0046] (1) Take the clinical biochemical complex quality control prepared by the present application from the packaging box, balance to room temperature (20-25℃), and open the quality control bottle cap to avoid loss of raw materials.

[0047] (2) Accurately take 5 ml of distilled water to reconstitute 1 bottle of quality control at room temperature of 20-25℃.

[0048] (3) Cover the rubber plug and tighten the bottle cap, and avoid light before use for 30 minutes.

[0049] (4) During reconstitution, gently rotate the vial several times to ensure complete dissolution of the contents.

[0050] (5) Before use, gently invert the vial to mix the contents. Do not shake the reagent bottle to avoid forming foam. Ensure that there is no freeze-dried material that has not been reconstituted.

[0051] Parameter setting: quality control reference value target value single, parameter setting see corresponding detection kit parameters.

[0052] Example 2

[0053] The difference between this example and example 1 is that human serum albumin is not added in the formula.

[0054] Example 3

[0055] The difference between this example and example 1 is that the complex enzyme stabilizer AES is not added in the formula.

[0056] Example 4

[0057] The difference between this example and Example 1 is that no freeze-drying step is performed after the sub-packaging, and the quality control product is in a liquid state.

[0058] Experimental results and data analysis

[0059] 1. Evaluation scheme

[0060] The freeze-dried powder of the composite quality control product obtained in Examples 1-4 was respectively evaluated for uniformity, thermal stability and long-term stability, wherein the composite quality control product freeze-dried powder was reconstituted in distilled water before measurement.

[0061] 2. Evaluation method

[0062] (1) Uniformity evaluation: The composite quality control product in each example was detected using the kit, and each example was detected 10 times to calculate the mean, standard deviation and coefficient of variation (CV), and the results are shown in Table 1.

[0063] (2) Thermal stability evaluation: The composite quality control product obtained in each example was stored at 37°C for 7 days, and the content of each enzyme was detected using the kit on the 0th day and the 7th day of storage, and each example was measured 3 times to calculate the average value, and the deviation of the measured value on the 7th day compared to the 0th day (%). The results are shown in Table 2.

[0064] (3) Long-term stability: The composite quality control product obtained in each example was stored at 2-8°C for 18 months, and the content of each enzyme in the composite quality control product was measured at 6, 12 and 18 months, and each example was measured 3 times to calculate the average value, and the deviation of each month compared to the 0th month (%). The results are shown in Table 3.

[0065] 3. Experimental results

[0066] Table 1: Uniformity data

[0067]

[0068]

[0069] Table 2: Thermal stability data

[0070]

[0071]

[0072] Table 3: Long-term stability data

[0073]

[0074]

[0075]

[0076] 4. Results analysis

[0077] According to the detection results of Tables 1-3, in general, the bias value of the quality control sample obtained by using the method described in Example 1 is relatively low after heat treatment at 37°C or long-term storage for 18 months, and the uniformity, thermal stability and long-term stability are all better, which can fully meet the use requirements.

[0078] Although the specific embodiments of the present application are described above, it is not a limitation on the scope of protection, and those skilled in the art should understand that various modifications or changes made by those skilled in the art on the basis of the technical solutions of the present application without creative labor are still within the scope of protection of the present application.

Claims

1. A method for preparing a clinical biochemical composite quality control product, characterized by: The composite quality control product comprises detection item raw materials, a matrix liquid and a protective agent; The detection item raw materials are: alkaline phosphatase 30-400 U / L, amylase 20-400 U / L, sodium glycocholate 2-80 μmol / L, cholinesterase 2000-13000 U / L, creatinine 40-300 μmol / L, leucine aminopeptidase 10-100 U / L, lactate dehydrogenase 50-500 U / L, lipase 20-100 U / L, triglyceride 0.4-5 mmol / L, cholesterol 3.12-10.4 mmol / L, uric acid 100-600 μmol / L, urea 1.43-20 mmol / L, alanine aminotransferase 7-100 U / L, aspartate aminotransferase 12-100 U / L, gamma-glutamyl transferase 10-100 U / L, glucose 2-10 mmol / L, alpha-hydroxybutyric acid dehydrogenase 30-300 U / L, sodium hydrogen phosphate 1-4 mg / dL, magnesium chloride 0.5-1.5 mg / dL, calcium chloride 2-3.75 mmol / L, ferrous chloride 6-50 μmol / L, bilirubin 2-100 μmol / L, pancreatic amylase 100-1000 U / L, sodium bicarbonate 10-40 mmol / L; The matrix liquid is: bovine serum albumin 30-90 g / L, human serum albumin 30-90 g / L; The protective agent is: dithiothreitol 1-5 g / L, mannitol 10-50 g / L, trehalose 10-50 g / L, sodium chloride 1-10 g / L, composite enzyme stabilizer AES 5-10 g / L, sodium azide 0.5 g / L; The preparation method of the clinical biochemical composite quality control product comprises the following steps: (1) The components are prepared according to the raw material formula and dissolved in distilled water in advance; (2) The bovine serum albumin and the human serum albumin are added under stirring and stirred until completely dissolved; (3) After the bovine serum albumin and the human serum albumin are completely dissolved, the solution is filtered into another clean preparation container by using a filter membrane; (4) The protective agent, including dithiothreitol, mannitol, trehalose, sodium chloride, composite enzyme stabilizer AES and sodium azide, is sequentially added into the solution in step (3) and stirred until completely dissolved; (5) After the substances in step (4) are completely dissolved, a basic stable solution of the composite quality control product is formed, which is filtered into another clean and sealable storage container by using a filter membrane and stored at 2-8 DEG C for standby; (6) The detection item raw materials are added into the solution in step (5) and stirred until uniformly mixed, so as to form a multi-item detection composite quality control product; (7) The multi-item detection composite quality control product obtained in step (6) is quantitatively packaged; (8) The packaged quality control product is subjected to freeze-drying operation to obtain a finished product, and the specific operation is that the product is pre-frozen for 4-5 h at a temperature of-40 DEG C, vacuumization is performed after the freeze-drying pressure reaches 150 ubar, and then drying is performed until the temperature of the freeze-drying machine reaches 25 DEG C, so as to obtain a freeze-dried powder of the clinical biochemical composite quality control product.

2. The method for preparing clinical biochemical composite quality control according to claim 1, characterized in that: The composite quality control product comprises detection item raw materials, a matrix liquid and a protective agent; The detection item raw materials are: alkaline phosphatase 350 U / L, amylase 350 U / L, sodium glycochenodeoxycholate 70 μmol / L, cholinesterase 10000 U / L, creatinine 200 μmol / L, leucine aminopeptidase 45 U / L, lactate dehydrogenase 420 U / L, lipase 80 U / L, triglyceride 1.5 mmol / L, cholesterol 7 mmol / L, uric acid 300 μmol / L, urea 15 mmol / L, alanine aminotransferase 50 U / L, aspartate aminotransferase 45 U / L, γ-glutamyl transferase 50 U / L, glucose 5 mmol / L, α-hydroxybutyric acid dehydrogenase 200 U / L, sodium hydrogen phosphate 4 mg / dL, magnesium chloride 1 mg / dL, calcium chloride 3 mmol / L, ferrous chloride 20 μmol / L, bilirubin 80 μmol / L, pancreatic amylase 500 U / L, sodium bicarbonate 20 mmol / L; The matrix liquid is: bovine serum albumin 30 g / L, human serum albumin 50 g / L; The protective agent is: dithiothreitol 3 g / L, mannitol 50 g / L, trehalose 50 g / L, sodium chloride 9 g / L, complex enzyme stabilizer AES 5 g / L, sodium azide 0.5 g / L.

3. The method for preparing clinical biochemical composite quality control according to claim 1, characterized in that: The stirring of the steps (2) and (4) is all at a speed of 400-500 r / min for 30-40 min until completely dissolved.

4. The method for preparing clinical biochemical composite quality control according to claim 1, characterized in that: The pore size of the filter membrane is 0.22 μm.

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