Preparation method of glycosylated hemoglobin standard substance in whole blood sample
Through specific dialysis and redissolution treatment, the problem of inconsistency between the standard substances of glycated hemoglobin and the matrix of the clinical sample is solved, and the good interchangeability of standard substances and clinical samples is achieved, and the difficulty of obtaining high-value samples is reduced.
Patent Information
- Application Number
- CN202411865510.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-04-18
AI Technical Summary
The existing standard glycated hemoglobin substances and clinical samples are not interchangeable due to matrix inconsistency, and high-value samples are difficult to obtain.
Low temperature dialysis is performed through a specific dialysis environment and dialysate to reduce the dissociation of bound glucose on HbA1c, using the buffer solution specified in the HbA1c reference method published by IFCC, and redissolve with ultrapure water or fetal bovine serum after lyophilization, ensuring that the matrix of the standard substance is consistent with the clinical sample.
The problem of inconsistency in the detection results caused by inconsistency between the standard substance and the matrix of the clinical sample was solved, and the difficulty of obtaining high-value HbA1c samples was reduced, achieving good interchangeability between the matrix of the standard substance and the clinical sample.
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Figure CN119845671A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a preparation and use method of a glycosylated hemoglobin standard substance in a whole blood sample, belonging to the technical field of biological sample measurement. Background Art
[0002] Glycated hemoglobin (HbA1c) testing is the golden indicator for blood sugar concentration management, diabetes diagnosis, and risk prediction of diabetes-related diseases. In order to achieve the standardization of glycated hemoglobin testing and the one-stop service between testing institutions, standardized management of test results is required. In 1995, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) established the HbA1c Standardization Working Group, and in 2002 officially published the international reference measurement system for HbA1c, and announced the reference measurement method for glycated hemoglobin and the standard substance for glycated hemoglobin. Because the plasma and pre-glycated hemoglobin in the whole blood sample interfere with the measurement of the reference measurement method, the HbA1c reference method published by IFCC requires the removal of plasma and pre-glycated hemoglobin by washing and incubating the fresh whole blood sample, and then adding a buffer of 2-morpholineethanesulfonic acid and sodium ethylenediaminetetraacetate to prepare the long-term storage sample to be tested. Glycated hemoglobin standard substances play an irreplaceable role in calibration or quality control in the clinical testing of this project.
[0003] At present, the standard materials at home and abroad are also prepared by referring to the sample processing method specified in the HbA1c reference method published by IFCC. Because the plasma part is removed from the standard materials prepared in this way, the HbA1c standard materials and clinical samples are not interchangeable when tested using clinical testing methods. In addition, due to the scarcity of blood samples, it is difficult to obtain high-value samples when preparing standard materials.
[0004] Therefore, this field needs to solve the problem of inconsistent matrices and lack of interchangeability between HbA1c standard substances and clinical samples when using clinical testing methods, as well as the problem of mismatched values; in addition, it is necessary to process normal concentration HbA1c samples into high-value HbA1c samples without changing the matrix composition of the standard substance, so as to reduce the difficulty of obtaining high-value raw materials for glycated hemoglobin standard substances. Summary of the invention
[0005] The first object of the present invention is to provide a method for preparing a glycated hemoglobin standard substance that is consistent with the clinical sample matrix, so as to solve the problem that the HbA1c standard substance and the clinical sample are not interchangeable due to the inconsistency of the matrix when the current clinical detection method is used for detection; the second object of the present invention is to process a normal concentration HbA1c sample into a high-value HbA1c sample without changing the matrix components of the standard substance, so as to reduce the difficulty of obtaining the raw materials of the glycated hemoglobin standard substance.
[0006] The technical solution of the present invention is as follows.
[0007] The present invention provides a method for preparing a glycated hemoglobin standard substance in a whole blood sample, comprising the following steps:
[0008] Step S1, centrifuging the whole blood sample separately and removing the upper plasma;
[0009] Step S2, adding equal volumes of sodium chloride solution to the samples obtained in step S1, mixing and centrifuging, and removing the upper layer of sodium chloride solution;
[0010] Step S3, repeating the operation in step S2 once or multiple times;
[0011] Step S4, adding cell incubation solution to the sample obtained in step S3 for incubation;
[0012] Step S5, centrifuging the sample obtained in step S4 to remove the cell incubation solution on the upper layer;
[0013] Step S6, freezing the sample obtained in step S5;
[0014] Step S7, re-melting the sample obtained in step S6, naturally breaking the cell wall, adding a buffer solution, shaking evenly, and measuring the initial glycated hemoglobin content;
[0015] Step S8, adding anhydrous glucose to the mixed sample obtained in step S7, stirring and incubating, and measuring the final glycated hemoglobin content;
[0016] Step S9, placing the sample obtained in step S8 in a dialysis container, placing a dialysate, a rotor and a dialysis bag containing the sample in the dialysis container; performing dialysis, repeating the dialysis multiple times, and terminating the dialysis when the glucose content of the sample is less than 6.0 mmol / L;
[0017] Step 10, packing the sample obtained in step S9 into ultrafiltration centrifuge tubes, and centrifuging to remove excess water in the sample;
[0018] Step S11, filtering the sample obtained in step S10 through a cell sieve and packaging it into multiple portions according to a certain volume; running the sample freeze-drying program until the water content in the sample is less than 3%, thereby obtaining a standard substance.
[0019] Preferably, the method further comprises re-dissolving the standard substance obtained in step S11 using a certain volume of solvent so that the total hemoglobin concentration in the re-dissolved sample is 100 g / L.
[0020] Preferably, the solvent is ultrapure water.
[0021] Preferably, the solvent is a biological sample that does not contain glycated hemoglobin and total hemoglobin.
[0022] Preferably, the biological sample is fetal bovine serum.
[0023] Preferably, the whole blood sample in step S1 is a human whole blood sample or other biological whole blood sample within 48 hours after blood collection.
[0024] Preferably, the sodium chloride solution in step S2 is physiological saline.
[0025] Preferably, the cell incubation solution in step S4 is a 0.1 mol / L to 0.2 mol / L sodium chloride solution.
[0026] Preferably, the dialysate in step S9 is a buffer solution of 10-100 mmol / l 2-morpholineethanesulfonic acid (MES) and 0.5-3 mmol / l sodium ethylenediaminetetraacetate (EDTA) with the pH adjusted to 5.5-7.5.
[0027] Preferably, the ambient temperature during dialysis in step S9 is 2-8° C.; the volume ratio of the dialysate to the sample is between 15:1 and 50:1.
[0028] Through the above technical solutions, the present invention can achieve the following technical effects.
[0029] The present invention utilizes a specific dialysis environment and dialysis fluid to solve the problem of instability of glycosylated hemoglobin in a sample during dialysis operation and the problem of inconsistency between the matrix of the prepared standard substance and the matrix of the measured sample.
[0030] The dialysis experiment in the present invention is carried out at 2-8°C. The glycosylation reaction of HbA1c is a reversible reaction. In the process of dialyzing the treated sample from high-concentration glucose to low-concentration glucose, part of the glucose bound to HbA1c will dissociate, resulting in a decrease in the glycosylated HbA1c value in the blood sample. The use of low-temperature dialysis at 2-8°C can greatly reduce the rate of dissociation of glucose bound to HbA1c. The matrix of the standard substance used for the evaluation of in vitro diagnostic reagents is required to be as consistent as possible with the matrix of the measured sample.
[0031] The dialysate used in the dialysis experiment of the present invention is a 50mmol / l 2-morpholineethanesulfonic acid (MES) and 1mmol / l sodium ethylenediaminetetraacetate (EDTA) buffer solution adjusted to pH 5.5-7.5. This buffer solution is the sample preservation solution specified in the HbA1c reference method published by IFCC. The sample treated with this dialysate does not add other reagents that may interfere with the detection of the clinical detection method, and also meets the requirements for assigning values to standard substances and standard products using the IFCC HbA1c reference method.
[0032] The present invention calculates the volume of ultrapure water required to achieve a total hemoglobin concentration of 100 g / L after the freeze-dried product is reconstituted, and then gives a definite value of this volume. This method avoids the cumbersome operation of adjusting the total hemoglobin concentration before freeze-drying.
[0033] The method of adjusting the total hemoglobin concentration in the sample and the technology of reconstituting the standard substance with fetal bovine serum after freeze-drying in the present invention solve the problem that after the standard substance is accurately assigned using the reference method, it is not interchangeable with the clinical sample when tested using the clinical detection method, and the problem of inconsistent measurement results.
[0034] The freeze-dried standard substance, after being reconstituted with ultrapure water, meets the sample pretreatment requirements of the HbA1c reference method published by IFCC, and can be accurately assigned a value by the reference method. The freeze-dried standard substance is reconstituted with a fixed volume of fetal bovine serum, which is consistent with the clinical sample matrix. It has been verified experimentally that this sample has good interchangeability with clinical samples when tested using clinical detection methods; this solves the problem that the existing HbA1c standard substance and clinical samples are not interchangeable due to inconsistent matrices. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 is a schematic diagram of the dialysis operation in the method of the present invention.
[0036] Figure 2 It is a schematic diagram of the re-dissolution operation of the standard substance prepared by the method of the present invention.
[0037] The meanings of the reference numerals in the figures are as follows:
[0038] T0, dialysis environment; T1, beaker; T2, dialysate; T3, dialysis bag; T4, sample; T5, magnetic stirring rotor; T6, constant temperature magnetic stirrer; T7, solvent; T8, vial; T9, standard substance. DETAILED DESCRIPTION
[0039] Example 1
[0040] like Figure 1-2 As shown, the preparation and use method of a glycated hemoglobin standard substance in a whole blood sample provided by the present invention includes the following steps.
[0041] Step S1, centrifuging 50 human whole blood samples within 48 hours after blood collection, and removing the upper plasma.
[0042] Step S2, adding an equal volume of physiological saline to the sample obtained in step S1, mixing and centrifuging, and removing the upper layer of physiological saline.
[0043] Step S3, repeat the operation in step S2 once.
[0044] Step S4, adding an equal volume of 0.15 mol / L cell incubation solution to the sample obtained in step S3, and incubating in a constant temperature incubator at 37° C. for 4 h.
[0045] Step S5, centrifuging the sample obtained in step S4 for 5 minutes to remove the cell incubation solution on the upper layer;
[0046] Step S6, freezing the sample obtained in step S5 at -70°C for more than 24 hours;
[0047] Step S7, reconstitute the samples obtained in step S6 at room temperature, break the cell walls naturally, add equal volumes of a buffer solution of 50 mmol / l 2-morpholineethanesulfonic acid (MES) and 1 mmol / l sodium ethylenediaminetetraacetate (EDTA) adjusted to pH 6.2, mix the obtained samples together, shake evenly, and measure the initial glycated hemoglobin content;
[0048] Step S8, anhydrous glucose is added to the mixed sample obtained in step S7 at a ratio of 1 g of anhydrous glucose to 1 ml of sample. A rotor is placed in the container of the mixed sample, and the container is placed in a 37°C constant temperature incubator, stirred and incubated on a constant temperature magnetic stirrer for 60 hours; and the final glycated hemoglobin content is measured.
[0049] Step S9, put the sample obtained in step S8 into a dialysis bag, put the dialysate, rotor and dialysis bag containing the sample into a beaker; put it into a constant temperature magnetic stirrer at 2-8°C, put the beaker on the constant temperature magnetic stirrer and perform dialysis under stirring, and replace the dialysate once every 8 hours; repeat the dialysis 4 times, and measure the glucose content in the sample with an automatic blood glucose analyzer to reduce to 5.2mmol / L; terminate the dialysis experiment, at which time the dialyzed sample swells due to water absorption.
[0050] Step S10, setting the refrigerated centrifuge to 2-8°C, placing the sample obtained in step S9 into 50KDa ultrafiltration centrifuge tubes, and centrifuging for 45 minutes to remove excess water from the sample;
[0051] Step S11, filter the sample obtained in step S10 through a 70 μm cell sieve, and dispense into vials at a volume of 0.4 ml per vial. Run the sample freeze-drying program on a vacuum freeze dryer until the water content in the sample is less than 3%;
[0052] Step S12, the standard substance prepared in step S11 is reconstituted with 0.4 ml ultrapure water, and the total hemoglobin concentration in the test sample is 79 g / L. After conversion, when the standard substance\standard product is reconstituted with 0.32 ml ultrapure water, the total hemoglobin concentration in the sample is 100 g / L.
[0053] Step S13, the standard substance prepared in step S12 is re-dissolved in 0.32 ml ultrapure water and then assigned a value using a reference method;
[0054] Step S14, the standard substance prepared in step S12 is reconstituted with 0.32 ml of fetal bovine serum and used for calibration or quality control of the standard substance\standard product in clinical test methods;
[0055] In step S9, the dialysate is a buffer solution containing 50 mmol / l 2-morpholineethanesulfonic acid (MES) and 1 mmol / l sodium ethylenediaminetetraacetate (EDTA), with the pH adjusted to 6.2. The ambient temperature during dialysis is 2-8°C.
[0056] Step S9 can be performed as follows: Figure 1 A sample dialysis method is shown, which is performed in a low-temperature dialysis environment T0 of 2-8°C, and the volume of the dialysate T2 and the sample T4 differs by 20 times. The beaker T1 is placed on a constant temperature magnetic stirrer T6, and a suitable magnetic stirring rotor T5 is placed in the beaker T1. The dialysate T2 is a buffer solution of 50mmol / l 2-morpholineethanesulfonic acid (MES) and 1mmol / l sodium ethylenediaminetetraacetate (EDTA) adjusted to pH 6.2, and the sample T4 is placed in a dialysis bag T3.
[0057] The dialysis environment T0 is a low temperature environment of 2-8°C. The main body T2 is a buffer solution of 50mmol / l 2-morpholineethanesulfonic acid (MES) and 1mmol / l sodium ethylenediaminetetraacetate (EDTA) adjusted to pH 6.2. The volume ratio of the dialysate T2 to the sample T4 should be between 20 times.
[0058] The dialysis process in step S9 is carried out at 2-8°C. The glycosylation reaction of HbA1c is a reversible reaction. During the dialyzing process of the treated sample from high-concentration glucose to low-concentration glucose, part of the glucose bound to HbA1c will dissociate, resulting in a decrease in the glycosylated HbA1c value in the blood sample. Using low-temperature dialysis conditions of 2-8°C can greatly reduce the rate at which glucose bound to HbA1c dissociates.
[0059] The matrix of the standard substance used for the evaluation of in vitro diagnostic reagents is required to be as consistent as possible with the matrix of the measured sample. The dialysate used in the dialysis experiment in step S9 is a 50mmol / l 2-morpholineethanesulfonic acid (MES) and 1mmol / l sodium ethylenediaminetetraacetate (EDTA) buffer solution with a pH adjusted to 5.5-7.5. This buffer solution is the sample preservation solution specified in the HbA1c reference method published by IFCC. The sample treated with this dialysate does not add other reagents that may interfere with the detection of the clinical detection method, and also meets the requirements for assigning values to standard substances and standard products using the IFCC HbA1c reference method.
[0060] Step S13 and step S14 can be implemented as follows: Figure 2 A method for re-dissolving a standard substance is shown, comprising the following steps.
[0061] The volume of ultrapure water / fetal bovine serum required to add to the standard substance is determined by calculating the volume of ultrapure water required to achieve a total hemoglobin concentration of 100 g / L after the freeze-dried product is reconstituted. In the process of assigning the value of the standard substance, the solvent T7 is ultrapure water or deionized water, and when the standard substance is tested using the clinical test method, the solvent T7 is fetal bovine serum.
[0062] The freeze-dried sample has different solvents T7 added according to different usage methods.
[0063] Those skilled in the art will appreciate that fetal bovine serum is selected in the present invention because it is stable and easy to obtain, and does not contain glycosylated hemoglobin and total hemoglobin. Other biological samples similar to fetal bovine serum may also be used.
[0064] Example 2
[0065] The present embodiment provides a method for preparing a glycated hemoglobin standard substance in a whole blood sample, comprising the following steps.
[0066] Step S1, centrifuging 20 human whole blood samples within 48 hours after blood collection, and removing the upper plasma.
[0067] Step S2, adding an equal volume of physiological saline to the sample obtained in step S1, mixing and centrifuging, and removing the upper layer of sodium chloride solution.
[0068] Step S3, repeat the operation in step S2 twice.
[0069] Step S4, adding an equal volume of 0.2 mol / L NaCl solution as an incubation solution to the sample obtained in step S3, and incubating at 39° C. in a constant temperature incubator for 4 h.
[0070] Step S5, centrifuging the sample obtained in step S4 for 5 minutes to remove the cell incubation solution on the upper layer.
[0071] Step S6, freezing the sample obtained in step S5 at -80°C for 20 hours.
[0072] Step S7, reconstitute the samples obtained in step S6 at room temperature, break the cell walls naturally, add equal volumes of a buffer solution of 50 mmol / l 2-morpholineethanesulfonic acid (MES) and 1 mmol / l sodium ethylenediaminetetraacetate (EDTA) adjusted to pH 6.5, mix the obtained samples together, shake evenly, and measure the initial glycated hemoglobin content.
[0073] Step S8, after filtering the sample obtained in step S10 through a 70 μm cell sieve, dispense it into vials with a volume of 0.4 ml per vial, and run a freeze-drying procedure on a vacuum freeze dryer until the water content in the sample is less than 3%.
[0074] Step S9: The standard substance prepared in step S11 is reconstituted with 0.4 ml ultrapure water, and the total hemoglobin concentration in the test sample is 66 g / L. After conversion, when the standard substance is reconstituted with 0.26 ml ultrapure water, the total hemoglobin concentration in the reconstituted sample is 100 g / L.
[0075] Step S10: The standard substance prepared in step S12 is reconstituted with 0.26 ml of ultrapure water and then assigned a value using the reference method.
[0076] In step S11 and step S12, the standard substance prepared is reconstituted with 0.26 ml of fetal bovine serum and used for calibration or quality control of the standard substance in clinical test methods.
[0077] Compared with Example 1, the incubation, dialysis and refrigerated centrifugation steps are omitted in this example, which simplifies the operation. However, the total hemoglobin concentration is low and it is only applicable to the situation where high-concentration glycated hemoglobin standard material does not need to be prepared.
[0078] Example 3
[0079] The specific method for preparing and using the glycated hemoglobin standard substance in the whole blood sample of this embodiment is as follows.
[0080] Instruments used:
[0081] 100,000-level analytical balance;
[0082] Constant temperature incubator;
[0083] Reagent refrigerator;
[0084] -70℃ refrigerator;
[0085] Refrigerated centrifuge;
[0086] Fully automatic blood glucose analyzer;
[0087] Constant temperature magnetic stirrer;
[0088] Vacuum freeze dryer;
[0089] pH meter;
[0090] 2000ml beaker;
[0091] 1000ml measuring cylinder;
[0092] 100ml blue cap reagent bottle;
[0093] 3500Da dialysis bag;
[0094] 5KDa ultrafiltration centrifuge tube;
[0095] 70μm cell sieve;
[0096] 1 box of dust-free paper (KIMTECH);
[0097] 2 pairs of powder-free latex gloves;
[0098] 1,000 straws;
[0099] Reagents used:
[0100] NaOH (Sigma);
[0101] NaCl (Sigma);
[0102] Anhydrous glucose (Sigma);
[0103] 2-Morpholineethanesulfonic acid (MES) (Sigma);
[0104] Sodium ethylenediaminetetraacetate (EDTA) (Sigma);
[0105] Sodium chloride injection;
[0106] Ultrapure water; in a preferred embodiment, prepared using a Milli-Q A10 ultrapure water machine;
[0107] Glycated hemoglobin determination kit (latex immunoturbidimetric method).
[0108] Balance room environment requirements:
[0109] The balance room should not be exposed to direct sunlight;
[0110] The balance workbench is firm and reliable, and the table surface is level;
[0111] The workroom should be clean and avoid the influence of airflow;
[0112] The temperature in the workroom should be constant;
[0113] It should be kept away from vibration sources, high-energy heat sources, high-intensity electromagnetic fields and other environments.
[0114] Experimental reagents and preparation:
[0115] Prepare cell incubation medium (0.15 mol / L NaCl solution): weigh 1.732 g NaCl on a balance, dissolve it in 100 ml ultrapure water, transfer it to a 200 ml volumetric flask, make up to volume, and shake well.
[0116] 4 mol / l sodium hydroxide solution: Weigh 32.0000 g of sodium hydroxide on a balance and dilute to 200 mL of ultrapure water.
[0117] Dialysate (50 mmol / l 2-morpholineethanesulfonic acid (MES) and 1 mmol / l sodium ethylenediaminetetraacetate (EDTA) buffer solution adjusted to pH 6.2): 17.66 g MES and 6.74 g EDTA were weighed on a balance and dissolved in 1800 mL water in a 2000 ml beaker. A pH meter was inserted and stirred on a constant temperature magnetic stirrer. The pH was adjusted to 6.2 with 4 mol / l sodium hydroxide.
[0118] Steps:
[0119] Step S1, number 40 human whole blood samples (about 1.5 ml each) within 48 hours after blood collection, Sam1 to Sam40 in sequence, and divide each of the samples numbered Sam31 to Sam40 into three equal portions, numbered Sam31a, Sam31b, Sam31c...Sam40a, Sam40b, Sam40c respectively.
[0120] Step S2, centrifuge 50 samples, namely Sam1-Sam30, Sam31a-Sam40a and Sam31b-Sam40b, for 3 minutes respectively, and remove the upper plasma with a pipette.
[0121] Step S3, adding an equal volume of sodium chloride injection to the sample obtained in step S2, mixing and centrifuging for 3 minutes, and removing the upper layer of sodium chloride injection with a pipette.
[0122] Step S4, repeat the operation in step S3 once.
[0123] Step S5, adding an equal volume of cell incubation solution to the sample obtained in step S4, and incubating in a constant temperature incubator at 37° C. for 4 hours.
[0124] Step S6, centrifuge the sample obtained in step S5 for 5 minutes, and remove the cell incubation solution on the upper layer with a pipette.
[0125] Step S7, freezing the sample obtained in step S6 in a -70°C refrigerator for 24 hours.
[0126] Step S8, reconstitute the samples obtained in step S7 at room temperature, break the cell walls naturally, and add equal volumes of 50mmol / l 2-morpholineethanesulfonic acid (MES) and 1mmol / l sodium ethylenediaminetetraacetate (EDTA) buffer solution adjusted to pH 6.2. Mix the samples numbered Sam1 to Sam30, transfer them into a 100ml blue-capped bottle, with a total volume of about 50ml, marked as RM, use a clinical test kit to detect the initial glycated hemoglobin content in RM to be 5.4%, and divide RM into two portions, each of about 25ml, numbered RM1 and RM2 respectively; at the same time, Sam31a to Sam40a and Sam31b to Sam40b are added with buffer and stored separately.
[0127] Step S9, add 25g of anhydrous glucose to the sample RM1, put a rotor in the blue-cap bottle containing the mixed sample, put the blue-cap bottle in a 37°C constant temperature incubator, stir and incubate on a constant temperature magnetic stirrer for 60 hours; use a clinical test kit to measure the final glycated hemoglobin content in the mixed sample to be 20.1%.
[0128] Step S10, placing the sample obtained in step S9 in a dialysis bag, placing the dialysate, rotor and dialysis bag containing the sample in a 2000ml beaker; placing a constant temperature magnetic stirrer in a 2-8°C reagent refrigerator, placing the 2000ml beaker on the constant temperature magnetic stirrer for dialysis under stirring, and replacing the dialysate once every 8 hours; repeating the dialysis 4 times, and measuring the glucose content in the sample with an automatic blood glucose analyzer to be 5.5mmol / L; terminating the dialysis experiment, and the dialyzed sample swells to about 100ml due to water absorption.
[0129] Step S11, setting the refrigerated centrifuge to 2-8°C, placing the sample obtained in step S10 into 50KDa ultrafiltration centrifuge tubes, and centrifuging for 45 minutes to remove excess water from the sample;
[0130] Step S12, filter the sample and RM2 obtained in step S11 through a 70 μm cell sieve, and dispense them into vials with a volume of 0.4 ml per vial. Run the sample freeze-drying procedure together with Sam31b to Sam40b on a vacuum freeze dryer until the water content in the sample is less than 3%;
[0131] Step S13, select one bottle of each level from the prepared RM1 and RM2, and reconstitute them with 0.4 ml ultrapure water respectively, and the total hemoglobin concentration in the test sample is 83 g / L and 80 g / L. After conversion, when reconstituted with 0.33 ml and 0.32 ml of ultrapure water respectively, the total hemoglobin concentration in RM1 and RM2 is guaranteed to be 100 g / L.
[0132] Step S14, select one bottle each from the packaged RM1 and RM2, use 0.33 ml and 0.32 ml of ultrapure water to reconstitute them respectively, and together with Sam31a~Sam40a, a total of 12 samples, are tested using the reference method. The results are shown in Table 1.
[0133] Step S15, select 2 bottles each from the packaged RM1 and RM2, and reconstitute them with 0.33 ml and 0.32 ml of fetal bovine serum and ultrapure water respectively. At the same time, Sam31b~Sam40b are reconstituted with 0.3 ml of fetal bovine serum respectively. Together with Sam31c~Sam40c, a total of 24 samples are tested using a glycated hemoglobin assay kit (latex immunoturbidimetry). The test results are shown in Table 1.
[0134] Table 1 Test results of samples under different conditions (unit: %)
[0135]
[0136] According to the experimental data, it can be concluded that the standard substance or sample prepared by the method before the present invention is reconstituted with ultrapure water, and the mean value of the clinical test method is about 6% lower than that of the reference method. When fetal bovine serum is used, most of the results of the clinical test method are significantly improved, and the mean value of the test results is basically consistent with the reference method. The method of reconstituted standard substances with fetal bovine serum solves the problem of inconsistent measurement results caused by the interchangeability of standard substances.
[0137] The present invention is described in detail above. Specific examples are used herein to illustrate the principle and implementation of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. For those skilled in the art, according to the idea of the present invention, there will be changes in the specific implementation and application scope. In summary, the content of this specification should not be understood as limiting the present invention.
Claims
1. A method for preparing a glycated hemoglobin standard substance in a whole blood sample, comprising the following steps: Step S1, centrifuging the whole blood sample separately and removing the upper plasma; Step S2, adding equal volumes of sodium chloride solution to the samples obtained in step S1, mixing and centrifuging, and removing the upper layer of sodium chloride solution; Step S3, repeating the operation in step S2 once or multiple times; Step S4, adding cell incubation solution to the sample obtained in step S3 for incubation; Step S5, centrifuging the sample obtained in step S4 to remove the cell incubation solution on the upper layer; Step S6, freezing the sample obtained in step S5; Step S7, re-melting the sample obtained in step S6, naturally breaking the cell wall, adding a buffer solution, shaking evenly, and measuring the initial glycated hemoglobin content; Step S8, adding anhydrous glucose to the mixed sample obtained in step S7, stirring and incubating, and measuring the final glycated hemoglobin content; Step S9, placing the sample obtained in step S8 into a dialysis container, placing a dialysate, a rotor and a dialysis bag containing the sample into the dialysis container; performing dialysis, repeating the dialysis multiple times, and terminating the dialysis when the glucose content of the sample is less than 6.0 mmol / L; Step 10, packing the sample obtained in step S9 into ultrafiltration centrifuge tubes, and centrifuging to remove excess water in the sample; Step S11, filtering the sample obtained in step S10 through a cell sieve and packaging it into multiple portions according to a certain volume; running the sample freeze-drying program until the water content in the sample is less than 3%, thereby obtaining a standard substance.
2. The method for preparing a glycated hemoglobin standard substance in a whole blood sample according to claim 1, characterized in that: The method further includes re-dissolving the standard substance obtained in step S11 using a certain volume of solvent so that the total hemoglobin concentration in the re-dissolved sample is 100 g / L.
3. The method for preparing a glycated hemoglobin standard substance in a whole blood sample according to claim 1, characterized in that: The solvent is ultrapure water.
4. The method for preparing a glycated hemoglobin standard substance in a whole blood sample according to claim 1, characterized in that: The solvent is a biological sample that does not contain glycated hemoglobin and total hemoglobin.
5. The method for using the glycated hemoglobin standard substance\standard product in a whole blood sample according to claim 4, characterized in that: The biological sample is fetal bovine serum.
6. The method for preparing a glycated hemoglobin standard substance in a whole blood sample according to claim 1, characterized in that: The whole blood sample in step S1 is a human whole blood sample or other biological whole blood sample within 48 hours after blood collection.
7. The method for preparing a glycated hemoglobin standard substance in a whole blood sample according to claim 1, characterized in that: The sodium chloride solution in step S2 is normal saline.
8. The method for preparing a glycated hemoglobin standard substance in a whole blood sample according to claim 1, characterized in that: The cell incubation solution in step S4 is a 0.1 mol / L to 0.2 mol / L sodium chloride solution.
9. The method for preparing a glycated hemoglobin standard substance in a whole blood sample according to claim 1, characterized in that: The dialysate in step S9 is a buffer solution of 10-100 mmol / l 2-morpholineethanesulfonic acid (MES) and 0.5-3 mmol / l sodium ethylenediaminetetraacetate (EDTA) with the pH adjusted to 5.5-7.
5.
10. The method for preparing a glycated hemoglobin standard substance in a whole blood sample according to claim 9, characterized in that: The ambient temperature during dialysis in step S9 is 2-8° C.; the volume ratio of the dialysate to the sample is between 15:1 and 50:1.