Glycosylated hemoglobin standard substance as well as preparation method and application thereof

Abstract

The invention discloses a glycosylated hemoglobin standard substance as well as a preparation method and application thereof. The preparation method of the glycosylated hemoglobin standard substance comprises the following steps: firstly, collecting a blood sample containing glycosylated hemoglobin, collecting red blood cells in the blood sample, and separating the lysed red blood cells to obtain hemoglobin; resuspending the hemoglobin in a phosphate buffer solution with the pH value of 8.4-8.6, and quantifying the concentration of the hemoglobin; and then adding a protein protection solution, uniformly mixing, sub-packaging to obtain the glycosylated hemoglobin standard substance, and finally determining the concentration of glycosylated hemoglobin. The standard substance prepared by the preparation method has good accuracy, uniformity, stability and interoperability, and can be used for traceability and quality evaluation of a glycosylated hemoglobin conventional detection system, preparation and quality evaluation of a glycosylated hemoglobin detection reagent, and preparation, valuing and quality evaluation of a calibrator and a quality control substance; and the stability is relatively high.

Description

Technical Field

[0001] The present invention relates to the field of medical testing technology, and in particular to a glycosylated hemoglobin standard substance and a preparation method and application thereof. Background Art

[0002] Diabetes is a major public health disease in the world and is showing an increasing trend. Diabetes is a metabolic disease characterized by high blood sugar. High blood sugar is caused by defects in insulin secretion or its biological function, or both. Long-term high blood sugar leads to chronic damage and dysfunction of various tissues, especially eyes, kidneys, heart, blood vessels, and nerves. It has become the third most serious chronic disease threatening human health after tumors and cardiovascular and cerebrovascular diseases.

[0003] Glycated hemoglobin is the product of the non-enzymatic reaction between hemoglobin in red blood cells and sugars (mainly glucose) in serum. The non-enzymatic reaction that forms glycated hemoglobin is continuous, slow, and irreversible. Therefore, the content of glycated hemoglobin is determined by the past rather than the immediate blood sugar concentration, and has nothing to do with factors such as whether the patient is fasting before the test, whether insulin is injected, and whether hypoglycemic drugs are taken. It is generally believed that the concentration of glycated hemoglobin can effectively reflect the average blood sugar level in the past 8-12 weeks. Glycated hemoglobin is composed of HbA1a, HbA1b, and HbA1c, of which HbA1c accounts for about 70% and has a relatively stable structure. It is often used as a monitoring indicator for diabetes control in clinical practice. The detection of HbA1c levels occupies an important position in clinical practice. According to the diabetes diagnosis guidelines, it has been included and has become an important indicator for diabetes diagnosis.

[0004] Conventional methods for clinical laboratory testing of glycated hemoglobin include: ion chromatography, affinity chromatography, electrophoresis, ion capture, chemiluminescence, immunoagglutination, isoelectric aggregation, enzyme method, etc. The different principles and methods used in the above detection methods lead to large differences in results between laboratories. Therefore, it is necessary to carry out standardization of glycated hemoglobin testing to ensure the traceability of the test results of each laboratory, and on this basis, achieve accuracy and comparability. The core of standardization work is to establish a reference system (including standard substances and reference methods). Standard substances are of great significance for achieving the traceability of the test results and ensuring the accuracy and comparability of the test results.

[0005] At present, the standard material of glycated hemoglobin can be stably stored for one year at -70°C. However, after low-temperature storage, freezing and thawing are generally only allowed once, and repeated freezing and thawing are not allowed. This is because slight environmental changes will affect the activity of the protein and the detection of glycated hemoglobin levels. In order to solve the above problems, the glycated hemoglobin standard material is packaged into small portions in this field, and one portion is used at a time. However, in the case of large inspection needs, a large number of small-package quality control products not only occupy a large amount of material resources and packaging work, but also occupy valuable storage space. Therefore, this field needs a glycated hemoglobin standard material that is easier to store and transport for use by clinical and testing institutions, and avoids artificial repeated freezing and thawing during transportation and use, thereby causing numerical changes. Summary of the invention

[0006] The present invention provides a glycated hemoglobin standard substance and a preparation method thereof, which are used to meet the requirements of clinical glycated hemoglobin standardization test.

[0007] In a first aspect, the present invention provides a method for preparing a glycated hemoglobin standard substance, comprising the following steps: Step 1, collecting a first blood sample and a second blood sample, wherein the first blood sample refers to a blood sample with a glycosylated hemoglobin concentration of 4.70%-5.70%, and the second blood sample refers to a blood sample with a glycosylated hemoglobin concentration of 9.60%-11.70%; Step 2: washing and lysing the red blood cells in the first blood sample and the second blood sample to obtain a first hemoglobin sample and a second hemoglobin sample; resuspending the first hemoglobin sample and the second hemoglobin sample in a phosphate buffer solution with a pH of 8.4-8.6, respectively, to obtain a phosphate buffer solution of the first hemoglobin sample and a phosphate buffer solution of the second hemoglobin sample; quantifying the concentration of hemoglobin in the phosphate buffer solution of the first hemoglobin sample and the phosphate buffer solution of the second hemoglobin sample; Step 3, adding a protein protection solution to the phosphate buffer solution of the first hemoglobin sample and the phosphate buffer solution of the second hemoglobin sample, respectively, and mixing them evenly, and packaging to obtain a first glycosylated hemoglobin standard substance and a second glycosylated hemoglobin standard substance; the protein protection solution comprises an antioxidant, a preservative and a protein freezing activity protective agent, and the pH of the protein protection solution is 8.0-8.8; Step 4: Determine the glycated hemoglobin concentrations in the first glycated hemoglobin standard substance and the second glycated hemoglobin standard substance by using isotope dilution mass spectrometry of peptide fragments cleaved by endoproteinase GLU-C.

[0008] In the above preparation method, the first blood sample and the second blood sample are derived from humans. Further, the first blood sample and the second blood sample are derived from healthy people, and the test results of hepatitis A virus antibody, hepatitis B surface antigen, hepatitis C antibody, hepatitis E virus antibody, gonococcal DNA determination, syphilis serum specific antibody and human immunodeficiency virus antibody are all negative.

[0009] In the above preparation method, the concentration of glycated hemoglobin in the first blood sample and the second blood sample can be obtained by conventional methods for detecting glycated hemoglobin in clinical laboratories, such as ion chromatography, affinity chromatography, electrophoresis, ion capture, chemiluminescence, immunoagglutination, isoelectric aggregation, enzyme method, etc. Since the concentration of the glycated hemoglobin standard substance should meet the needs of clinical testing, that is, to meet the detection of people with low levels of glycated hemoglobin and people with high levels of glycated hemoglobin, the first blood sample contains a lower concentration of glycated hemoglobin, specifically a blood sample with a glycated hemoglobin concentration of 4.70%-5.70%, and the second blood sample contains a higher concentration of glycated hemoglobin, specifically a blood sample with a glycated hemoglobin concentration of 9.60%-11.70%.

[0010] It should be noted that the concentration of glycated hemoglobin in the first blood sample and the second blood sample only needs to be within the above range, and may include blood samples from different individuals, that is, the first blood sample and the second blood sample are obtained by mixing blood samples from different individuals.

[0011] In the above preparation method, in step 2, the first blood sample and the second blood sample are first centrifuged at a speed of 2000-3000rpm / min for 5-10min, respectively, and the red blood cells are collected after removing the supernatant, and the red blood cells are washed 2-3 times with phosphate buffer. Next, the collected red blood cells are lysed to release the hemoglobin therein; specifically, a hypotonic lysis method can be used, in which the red blood cells are placed in a hypotonic solution, such as distilled water, and the red blood cells absorb water and swell and rupture, releasing the hemoglobin therein. Finally, the substances collected after the red blood cell lysis are removed from impurities to obtain the first hemoglobin sample and the second hemoglobin sample.

[0012] In the above preparation method, the concentration of hemoglobin can be detected by cyanomethemoglobin (HiCN) colorimetric method.

[0013] In the above-mentioned preparation method, the protein protective solution is used to ensure the stability of the glycated hemoglobin standard material. For different samples and different biomolecules, the effects of different protein protective solutions vary greatly. Taking glycated hemoglobin as an example, although glycated hemoglobin can be stored for a long time at low temperatures, for glycated hemoglobin standard materials, minor changes (e.g., protein denaturation and decreased activity) will affect the detection of glycated hemoglobin levels in the sample. Therefore, glycated hemoglobin standard materials are generally only allowed to be frozen and thawed once after low temperature storage, and repeated freezing and thawing is not allowed to avoid repeated freezing and thawing causing a decrease in the quality of the standard. In order to solve the above problems, the protein protective solution provided by the present invention includes an antioxidant, a preservative and a protein freezing activity protective agent.

[0014] Furthermore, the antioxidant is selected from at least one of tert-butylhydroquinone, dithiothreitol, β-mercaptoethanol, ascorbyl palmitate, phytic acid, and tert-butylhydroxyanisole.

[0015] Furthermore, the preservative is selected from at least one of benzimidazole, hydroxybenzoic acid ester and ethylene glycol phenyl ether.

[0016] Further, the protein cryoprotectant is a class of compounds used to protect proteins from losing activity during freezing and thawing, and they protect the structure and function of proteins by preventing protein denaturation, aggregation and the formation of ice crystals. However, for proteins, good protein cryoprotectants generally include glycerol, amino acids, surfactants, proteins (e.g., albumin), synthetic polymers (e.g., polyethylene glycol, PVP), disaccharides, etc. The protein cryoprotectant used in the present invention is selected from at least one of inulin, dextran, inositol, and raffinose. The present invention selects carbohydrate compounds as protein cryoprotectants, which can avoid the negative effects of some cryoprotectants on various detection methods. For example, in HPLC, glycerol may affect the retention time and peak shape of the analyte; in affinity chromatography, glycerol may affect the binding and elution process; polyethylene glycol is used as a co-solvent in the detection of glycated hemoglobin by ion exchange chromatography, which will affect the chromatographic behavior of hemoglobin; certain amino acids such as glycine and glutamic acid may interfere with chemical reactions or spectral detection, thereby affecting the accuracy of detection; and certain salt protective agents may cause changes in glycated hemoglobin standard substances during repeated freezing and thawing, resulting in a decrease in quality and failure to meet quality control requirements. The protein cryoprotectant provided by the present invention helps to ensure the stability of glycated hemoglobin standard substances under repeated freezing and thawing conditions.

[0017] Furthermore, the solvent of the protein protection solution may be a phosphate buffer solution.

[0018] Furthermore, the antioxidant is preferably tert-butyl hydroquinone and tert-butyl hydroxyanisole. The preservative is preferably ethylene glycol phenyl ether. The protein freezing activity protective agent is preferably inulin, dextran and raffinose.

[0019] Furthermore, in the protein protection solution, the concentration of the antioxidant is 0.01-0.05 g / L, the concentration of the preservative is 0.01-0.2 g / L, and the concentration of the protein freezing activity protective agent is 200-300 g / L.

[0020] Furthermore, the volume ratio of the phosphate buffer solution to the protein protection solution of the first hemoglobin sample is 1:(30-60), and the volume ratio of the phosphate buffer solution to the protein protection solution of the second hemoglobin sample is 1:(30-60).

[0021] In the above preparation method, the concentration of glycated hemoglobin in the first glycated hemoglobin standard substance is 5.36%, and when the coverage factor k=2, the uncertainty of the concentration is 0.31%; In the second glycated hemoglobin standard substance, the concentration value of glycated hemoglobin is 10.66%. When the coverage factor k=2, the uncertainty of the concentration value is 0.61%.

[0022] The above-mentioned concentration of glycated hemoglobin is expressed as a percentage of hemoglobin.

[0023] In the above preparation method, the method also includes performing uniformity evaluation, stability evaluation, uncertainty evaluation and interoperability evaluation on the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance with fixed values.

[0024] In the above preparation method, in order to ensure the stability of hemoglobin, the temperature of the mixed system is controlled at 2°C-8°C during the preparation process.

[0025] In a second aspect, the present invention provides a first glycosylated hemoglobin standard substance and / or a second glycosylated hemoglobin standard substance prepared by the above preparation method.

[0026] In a third aspect, the present invention provides the use of the first glycated hemoglobin standard substance and / or the second glycated hemoglobin standard substance in the preparation of clinical diagnostic reagents, calibrators, and quality control materials for detecting glycated hemoglobin, traceability of measurement values, and quality evaluation analysis.

[0027] In a fourth aspect, the present invention provides a protein protection solution, comprising an antioxidant, a preservative, a protein freezing activity protection agent, and a pH buffer; The pH of the protein protection solution is 8.0-8.8.

[0028] In the above-mentioned protein protection solution, the antioxidant is selected from at least one of tert-butylhydroquinone, dithiothreitol, β-mercaptoethanol, ascorbyl palmitate, inositol hexaphosphate, and tert-butylhydroxyanisole; the preservative is selected from at least one of benzimidazole, hydroxybenzoic acid esters, and ethylene glycol phenyl ether; and the protein freezing activity protective agent is selected from at least one of inulin, dextran, inositol, and raffinose.

[0029] Compared with the prior art, the present invention has the following advantages: 1. The glycated hemoglobin standard material provided by the present invention has good uniformity, stability and interoperability, and meets the requirements of international standardization work, the "Technical Specifications for Primary Standard Materials" and ISO guide 35. 2. The glycated hemoglobin standard substance provided by the present invention has good stability. It can be stably stored at 2-8°C for 5 days, can be stably stored at -20°C for 12 months, and can be long-term stored at -70°C for 24 months. It also has good stability under re-thawing conditions. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0031] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0032] Example 1 This embodiment provides a method for preparing a glycated hemoglobin standard substance, comprising the following steps: (1) Preparation of healthy human blood by centrifugation: Taking a first blood sample with a glycated hemoglobin concentration of 4.70%-5.70% and a second blood sample with a glycated hemoglobin concentration of 9.60%-11.70%, centrifuging the first blood sample and the second blood sample at 2000-3000 rpm / min for 5-10 min, respectively, removing serum from the blood, collecting the precipitate, washing the precipitate twice with phosphate buffer, hypotonic lysing red blood cells, and centrifuging to obtain the first hemoglobin and the second hemoglobin; (2) Preparation of protein protection solution: Add 0.01 g / L tert-butylhydroquinone, 0.01 g / L tert-butylhydroxyanisole, 0.1 g / L ethylene glycol phenyl ether, 25.0 g / L inulin, 75.0 g / L dextran and 150.0 g / L raffinose to phosphate buffer solution, adjust the pH value of the solution to 8.0-8.8, and mix well to prepare protein protection solution; (3) Preparation of glycated hemoglobin standard substance: Add pH=8.5 phosphate buffer to the first hemoglobin and the second hemoglobin obtained in step (1), respectively, and mix by pipetting to obtain a phosphate buffer solution of the first hemoglobin sample and a phosphate buffer solution of the second hemoglobin sample; The concentrations of hemoglobin in the phosphate buffer solution of the quantitative first hemoglobin sample and the phosphate buffer solution of the second hemoglobin sample are both 55 g / L. Take 1 ml of the phosphate buffer solution of the first hemoglobin sample and 1 ml of the phosphate buffer solution of the second hemoglobin sample respectively and mix them with 49 ml of the protective solution by inverting (the solution temperature is controlled at 2°C-8°C during the preparation process to avoid protein degradation).

[0033] (4) Packaging and storage of glycated hemoglobin standard substance: Use a pipette to dispense 100 μL of the mixed solution in step (3) into a brown vial, seal it with a cap, and store it in an ultra-low temperature refrigerator at -70°C to obtain a first glycated hemoglobin standard substance and a second glycated hemoglobin standard substance.

[0034] (5) Glycated hemoglobin reference material value determination: 1. Pretreatment of standard substance samples: (I) Take 0.1 ml of glycosylated hemoglobin standard substance and add 0.1 ml of urea solution (16.0 mol / L) to make the urea concentration of the reaction solution 8.0 mol / L; (II) 100 μg of glycosylated hemoglobin was reduced by adding 5.0 mmol / L dithiothreitol at 37°C for 1 hour; (III) adding iodoacetic acid in 1.0 mol / L sodium hydroxide to a final concentration of 14.0 mmol / L, and carrying out the alkylation reaction for 30 min at room temperature in the dark; (IV) Add 5.0 mmol / L dithiothreitol at room temperature for 1 hour to terminate the alkylation reaction; (V) diluting with 50.0mmol / L Tris-HCl solution to make the final urea concentration 1.5mol / L; (VI) adding endoproteinase GLU-C and digesting at 37°C overnight; (VII) Add 10% formic acid solution to terminate the digestion reaction; (VIII) The digested peptides are dried under vacuum centrifugation; (IX) Add 0.1% formic acid solution to the required sample concentration and take the supernatant for analysis.

[0035] 2. When pre-treating the glycated hemoglobin standard substance, add a deuterium isotope labeled specific peptide (amino acid sequence: Val-His-Leu-Thr-Pro-Glu) as an internal reference.

[0036] 3. High performance liquid chromatography separation: (I) Equipment and reagent information, see Table 1.

[0037] Table 1 Equipment and reagent information

[0038] (II) Separation and elution: injection volume was 10.0 μL, flow rate was 0.30 mL / min, and specific conditions were shown in Table 2.

[0039] Table 2 HPLC separation and elution conditions

[0040] 4. Mass spectrometer detection: (I) Equipment information: Triple quadrupole-ion trap mass spectrometer (model: Qtrap4000, AB SCIEX, USA) using electrospray ionization (ESI) source and positive ion multiple reaction monitoring (MRM) mode; injection volume: 1.0 μL.

[0041] (II) Equipment operating parameters, see Table 3 for details.

[0042] Table 3 Mass spectrometer operating parameters

[0043] 5. Standard substance sample mean value detection Using the above equipment, 50 cases / level were randomly selected; samples of each level were tested: 10 bottles / day, 1 time / bottle, and the test results were recorded for 5 consecutive days.

[0044] 6. Test results (mean ± standard deviation) SPSS16.0 was used to analyze the overall mean and standard deviation of the data. The concentration results of the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance are as follows: (1) The first glycosylated hemoglobin standard substance: 5.36% ± 0.31%; (2) The second glycosylated hemoglobin standard substance: 10.66%±0.61%.

[0045] Example 2 Homogeneity test of glycosylated hemoglobin standard substance (1) Random sampling of reference materials: Using the above equipment and detection method, 15 bottles of the minimum packaging units of the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance were randomly selected and randomly numbered after sampling.

[0046] (2) Standard substance testing: The above measuring equipment is used to test the random samples, and each sample is tested 3 times; the test order is: 1, 3, 5, 7, 9, 11, 13, 15, 2, 4, 6, 8, 10, 12, 14, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 12, 14, 1, 3, 5, 7, 9, 11, 13, 15, and the data results are recorded.

[0047] (3) Statistical analysis: The above data were statistically analyzed using the following formula:

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] In the above formula, represents the variance, Indicates the i-th measured value or calculated result of the specified parameter. represents the overall mean, represents the number of repeated measurements of sample i, represents the jth measured value of sample i, represents mean square, represents the degrees of freedom, represents the test value, Indicates the number of valid tests, represents the number of samples drawn, Indicates the total number of tests. represents the standard deviation between bottles, represents the within-bottle standard deviation.

[0060] When the statistical result F≤10, it is considered that the uniformity between standard bottles is good and it can be used as a standard material. The average value of all test results is calculated, and then the calculation between cv bottles and within cv bottles is performed; when the statistical result F>10, it is considered that the uniformity between standard bottles is poor and it is not suitable to be used as a standard material. At the same time, the calculation between cv bottles and within cv bottles is no longer performed; when the statistical result F≤1, the standard deviation within the bottle is used instead of the standard deviation between bottles. = .

[0061] (4) Uniformity results: The first glycosylated hemoglobin standard material: F = 1.43, cv between bottles = 0.28%, cv within bottles = 0.73%; The second glycosylated hemoglobin standard substance: F=0.78, cv between bottles=0.46%, cv within bottle=0.46%.

[0062] The above experiments prove that the glycated hemoglobin standard substance prepared by the present invention has good uniformity and meets the qualified requirements of the standard substance.

[0063] Example 4 Study on the stability of glycated hemoglobin standard substance (1) Research design (I) Temperature design: 2℃-8℃, -20℃, -70℃, 20℃-30℃, -20℃ to -30℃.

[0064] (II) Time point design, see Table 4 for details.

[0065] Table 4 Stability test design

[0066] Note: In Table 4, 2℃-8℃ short-term stability refers to placing the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance at 2℃-8℃, and testing the glycosylated hemoglobin concentration on days 1-5; 2℃-8℃ open bottle stability refers to placing the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance in a 2℃-8℃ refrigerator on days 5, 4, 3, 2, 1 and 0 before the test, and testing the glycosylated hemoglobin concentration on day 0; 2℃-8℃ reconstitution stability refers to It means that on the 5th, 4th, 3rd, 2nd, 1st and 0th day before the test, the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance are re-thawed and placed in a 2℃-8℃ refrigerator, and the glycosylated hemoglobin concentration is tested on the 0th day; 2℃-8℃ transportation stability means that the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance are transported at 2℃-8℃, and the glycosylated hemoglobin standard substances are tested on each day on the 1st, 2nd, 3rd, 4th and 5th day; 20℃-30℃ short-term stability means that the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance are transported at 2℃-8℃, and the glycosylated hemoglobin standard substances are tested on each day; The first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance are placed at 20℃-30℃, and the glycosylated hemoglobin concentration is tested at 1 hour, 3 hours, 5 hours, 8 hours, 12 hours and 24 hours after being placed in the condition; the -20℃ long-term stability is to place the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance at -20℃, and then test the glycosylated hemoglobin concentration at 1 month, 2 months, 3 months, 5 months, 9 months and 12 months after being placed in the condition. Test; -70℃ long-term stability means placing the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance at -70℃, and then testing the glycosylated hemoglobin concentration at 1 month, 3 months, 5 months, 12 months, 18 months and 24 months after that; -30℃ to -20℃ repeated freeze-thaw stability means freezing and thawing the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance stored at -30℃ to -20℃ for 3 times, and then testing the glycosylated hemoglobin concentration.

[0067] (2) Inspection method The short-term stability was tested three times per bottle using the HLC-723G8 fully automatic glycated hemoglobin analyzer produced by TOSOH CORPORATION; the long-term stability was tested three times per bottle using the above-mentioned high performance liquid mass spectrometer; the glycated hemoglobin standard substance was stored at -70°C as the "zero point" for each stability test.

[0068] (3) Statistical analysis Perform linear regression analysis on the mean of the test data and calculate the slope and the intercept estimate , the formula is as follows:

[0069]

[0070]

[0071]

[0072]

[0073] In the above formula, represents the uncertainty introduced by remelting, Indicates the time point. represents the uncertainty of the slope, represents the slope, represents the intercept, represents the standard deviation of the straight line, Indicates a point in time, represents the mean value at a time point, represents the mean of the test results at a time point, Represents the grand average of the test results at a time point.

[0074] (4) Eligibility criteria:

[0075] (5) Test results, see Table 5.

[0076] Table 5 Stability test results

[0077] The above experiments prove that the glycated hemoglobin standard substance prepared by the present invention has good long-term, bottle opening, re-melting, short-term and transportation stability, meets the qualified requirements of standard substances, and has better transportation stability.

[0078] Example 5 Uncertainty Assessment of Glycated Hemoglobin Standard Material 1. Analysis of sources of uncertainty Refer to "Evaluation of Measurement Uncertainty of Reference Materials for In Vitro Diagnostic Reagents" YY / T 1709-2020; the uncertainty introduced by glycated hemoglobin standard materials includes: fixed value: measurement precision, bias and working glycated hemoglobin standard materials; uniformity; stability: opening stability, reconstitution stability and shelf life stability.

[0079] 2. Sampling of reference materials Sampling of glycosylated hemoglobin standard substances: Randomly select 100 bottles of the first glycosylated hemoglobin standard substance and 100 bottles of the second glycosylated hemoglobin standard substance.

[0080] 3. Measurement procedures High performance liquid chromatography-mass spectrometry was used to analyze the peptide fragments cleaved by endoproteinase GLU-C using isotope dilution mass spectrometry.

[0081] 4. Uncertainty assessment method (1) Measurement precision The above measurement procedure was used to test 50 bottles of the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance randomly selected: 10 bottles / day, 3 times / bottle, and the test results were recorded for 5 consecutive days. The uncertainty analysis of the data was performed using the following formula:

[0082]

[0083] In the above formula, represents the overall mean, Indicates the precision uncertainty.

[0084] (2) Bias The above measurement procedure was used to calibrate the radioactive deuterium labeled standard material, and the glycated hemoglobin standard material was tested. The measurement was repeated 5 times, and the uncertainty analysis was performed using the following formula:

[0085] In the above formula, represents the standard error of the mean of multiple measurements, Indicates the standard uncertainty of the stated value.

[0086] (3) Working glycosylated hemoglobin standard material Check the reference material certificates of leucine deuterium isotope label, threonine deuterium isotope label and valine deuterium isotope label. The uncertainty introduced by the above reference materials is calculated using the following formula:

[0087] In the above formula, Indicates the working glycated hemoglobin standard material value, represents the expanded uncertainty of the fixed value, Denotes the corresponding coverage factor.

[0088] (4) Uniformity The above measurement procedure was used to test 15 bottles of the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance which were randomly selected and numbered; the test order was: 1, 3, 5, 7, 9, 11, 13, 15, 2, 4, 6, 8, 10, 12, 14, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 12, 14, 1, 3, 5, 7, 9, 11, 13, 15; the data results were recorded and the uncertainty analysis was performed using the following formula:

[0089] In the above formula, represents the test mean of bottle i, represents the number of repeated tests of bottle i, represents the mean square between bottles, represents the mean square in the bottle, Represents the uncertainty of uniformity.

[0090] (5) Opening stability Randomly select 12 bottles of the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance. Open the bottles of glycosylated hemoglobin standard substance on the 5th, 4th, 3rd, 2nd, 1st and 0th days before the test (2 bottles / day) and place them in a 2℃-8℃ refrigerator. On the 0th day, repeat the measurement of glycosylated hemoglobin standard substance 3 times / bottle using the above measurement procedure, record the data results, and use the following formula to calculate the uncertainty:

[0091]

[0092]

[0093]

[0094]

[0095] In the above formula, represents the uncertainty introduced by opening the bottle, Indicates the time point. represents the uncertainty of the slope, represents the slope, represents the intercept, represents the standard deviation of the straight line, Indicates a point in time, represents the mean value at a time point, represents the mean of the test results at a time point, Represents the grand average of the test results at a time point.

[0096] (6) Remelting stability Randomly select 12 bottles of the first glycosylated hemoglobin standard substance and the second glycosylated hemoglobin standard substance. On the 5th, 4th, 3rd, 2nd, 1st and 0th days before the test, re-thaw the glycosylated hemoglobin standard substance and place it in a 2℃-8℃ refrigerator. On the 0th day, repeat the measurement of the glycosylated hemoglobin standard substance 3 times / bottle using the above measurement procedure, record the data results, and use the following formula to calculate the uncertainty:

[0097]

[0098]

[0099]

[0100]

[0101] In the above formula, represents the uncertainty introduced by remelting, Indicates the time point. represents the uncertainty of the slope, represents the slope, represents the intercept, represents the standard deviation of the straight line, Indicates a point in time, represents the mean value at a time point, represents the mean of the test results at a time point, Represents the grand average of the test results at a time point.

[0102] (7) Stability during shelf life The uncertainty introduced by the validity period has been verified to be less than 1 / 3 of the maximum uncertainty and is therefore not included in the total uncertainty.

[0103] (8) Uncertainty introduced by fixed value:

[0104]

[0105] In the above formula, It means that the fixed value introduces relative uncertainty. represents the introduced standard uncertainty, Indicates the product's glycated hemoglobin standard substance value.

[0106] (9) Uncertainty synthesis a. Combined standard uncertainty:

[0107] b. Synthetic expanded uncertainty:

[0108] In the above formula, k represents the inclusion factor, k =2.

[0109] (10) Determination of the value of glycated hemoglobin reference material (1) Level 1: 5.36% ± 0.31%, k =2; (2) Level 2: 10.66% ± 0.61%, k =2.

[0110] The above experiments can prove that the uncertainty of the glycated hemoglobin standard prepared by the present invention is good and meets the qualified requirements of the standard material.

[0111] Example 6 Interoperability test of the glycated hemoglobin standard substance 1. Matrix effect based on interoperability evaluation design Refer to the requirements of WS / T356-2011 "Guidelines for Matrix Effect and Interoperability Evaluation"; Detection methodologies include: chemiluminescence, high performance liquid chromatography, immunoturbidimetry and enzymatic methods.

[0112] (II) Testing materials (1) Standard materials: Take 3 bottles of the first glycosylated hemoglobin standard material and 3 bottles of the second glycosylated hemoglobin standard material. The first glycosylated hemoglobin standard materials are numbered LRM1-LRM3; the second glycosylated hemoglobin standard materials are numbered HRM1-HRM3; (2) Clinical samples: 20 fresh blood samples (hemoglobin concentration range: 4.5%-12%) and randomly numbered S1-S20.

[0113] (III) Detection methods The testing was performed according to the instruction manual of each testing equipment and the laboratory operating procedures. The testing order of each sample is shown in Table 6, and each sample was tested three times.

[0114] Table 6 Test sequence of standard substances and blood samples

[0115] (IV) Test results (1) High performance liquid chromatography, as shown in Table 7.

[0116] Table 7 HPLC test results for individual samples

[0117] (2) Chemiluminescence detection results are shown in Table 8.

[0118] Table 8 Chemiluminescence test results for each sample

[0119] (3) The results of immunoturbidimetric assay are shown in Table 9.

[0120] Table 9 Test results of each sample by immunoturbidimetry

[0121] (4) Enzymatic test results are shown in Table 10.

[0122] Table 10 Enzymatic method test results of each sample

[0123] (V) Data Statistical Analysis (1) Outlier check: Outlier analysis was performed on the above data and no outlier data was found.

[0124] (2) Regression analysis: The above data were analyzed by linear regression. The linear regression equation was obtained based on the above 20 blood test data. Substituting the value of the standard substance into the above equation, the predicted value of the sample can be obtained.

[0125] (3) Calculate the expected interval and compare the predicted value with the measured value. The test results of each test method are shown in Tables 11-13.

[0126] Table 11 Comparison of results between chemiluminescence and HPLC

[0127] Table 12 Comparison of results between immunoturbidimetry and HPLC

[0128] Table 13 Comparison of results between enzymatic method and HPLC method

[0129] (VI) Results Analysis In the detection methods based on different principles, such as chemiluminescence, high performance liquid chromatography, immunoturbidimetry and enzyme method, all the test results were within the range of 95% prediction upper limit and 95% prediction lower limit, and the matrix of glycated hemoglobin had no effect on the test results.

[0130] The above experiments can also prove that the glycated hemoglobin standard substance prepared by the present invention has no matrix effect and good intercommunication, and meets the qualified requirements of the standard substance.

[0131] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a glycated hemoglobin standard substance, characterized in that: The steps include: Step 1, collecting a first blood sample and a second blood sample, wherein the first blood sample refers to a blood sample with a glycosylated hemoglobin concentration of 4.70%-5.70%, and the second blood sample refers to a blood sample with a glycosylated hemoglobin concentration of 9.60%-11.70%; Step 2: washing and lysing the red blood cells in the first blood sample and the second blood sample to obtain a first hemoglobin sample and a second hemoglobin sample; resuspending the first hemoglobin sample and the second hemoglobin sample in a phosphate buffer solution with a pH of 8.4-8.6, respectively, to obtain a phosphate buffer solution of the first hemoglobin sample and a phosphate buffer solution of the second hemoglobin sample; quantifying the concentration of hemoglobin in the phosphate buffered solution of the first hemoglobin sample and the phosphate buffered solution of the second hemoglobin sample; Step 3, adding a protein protection solution to the phosphate buffer solution of the first hemoglobin sample and the phosphate buffer solution of the second hemoglobin sample, respectively, and mixing them evenly, and packaging to obtain a first glycosylated hemoglobin standard substance and a second glycosylated hemoglobin standard substance; the protein protection solution comprises an antioxidant, a preservative and a protein freezing activity protective agent, and the pH of the protein protection solution is 8.0-8.8; Step 4: Determine the glycated hemoglobin concentrations in the first glycated hemoglobin standard substance and the second glycated hemoglobin standard substance by using isotope dilution mass spectrometry of peptide fragments cleaved by endoproteinase GLU-C.

2. The preparation method according to claim 1, characterized in that: Step 2 also includes: centrifuging the first blood sample and the second blood sample at a speed of 2000-3000 rpm / min for 5-10 minutes respectively, removing the supernatant and collecting the red blood cells, and washing the red blood cells 2-3 times with phosphate buffer.

3. The preparation method according to claim 1 or 2, characterized in that: The antioxidant is selected from at least one of tert-butylhydroquinone, dithiothreitol, β-mercaptoethanol, ascorbyl palmitate, phytic acid, and tert-butylhydroxyanisole; And / or, the preservative is selected from at least one of benzimidazole, hydroxybenzoic acid ester, and ethylene glycol phenyl ether; And / or, the protein freezing activity protective agent is selected from at least one of inulin, dextran, inositol and raffinose.

4. The preparation method according to any one of claims 1 to 3, characterized in that In the first glycated hemoglobin standard substance, the concentration value of glycated hemoglobin is 5.36%, and when the coverage factor k=2, the uncertainty of the concentration value is 0.31%; In the second glycosylated hemoglobin standard substance, the concentration value of glycosylated hemoglobin is 10.66%. When the coverage factor k=2, the uncertainty of the concentration value is 0.61%.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The method also includes performing uniformity evaluation, stability evaluation, uncertainty evaluation and interoperability evaluation on the first glycosylated hemoglobin standard material and the second glycosylated hemoglobin standard material with fixed values.

6. The first glycosylated hemoglobin standard substance and / or the second glycosylated hemoglobin standard substance prepared according to the preparation method according to any one of claims 1 to 5.

7. Use of the first glycosylated hemoglobin standard substance and / or the second glycosylated hemoglobin standard substance according to claim 6 in the preparation of clinical diagnostic reagents, calibrators, and quality control materials for detecting glycosylated hemoglobin, value traceability, and quality evaluation analysis.

8. A protein protection solution, characterized in that: including antioxidants, preservatives, protein cryoprotectants and pH buffers; The pH of the protein protection solution is 8.0-8.

8.

9. The protein protection solution according to claim 8, characterized in that: The antioxidant is selected from at least one of tert-butylhydroquinone, dithiothreitol, β-mercaptoethanol, ascorbyl palmitate, phytic acid, and tert-butylhydroxyanisole; And / or, the preservative is selected from at least one of benzimidazole, hydroxybenzoic acid ester, and ethylene glycol phenyl ether; And / or, the protein freezing activity protective agent is selected from at least one of inulin, dextran, inositol and raffinose.

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