CN115372631A - Kit for preparing glycosylated hemoglobin calibrator and preparation method - Google Patents
Kit for preparing glycosylated hemoglobin calibrator and preparation method Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
Abstract
The application relates to the technical field of biology, and particularly discloses a kit for preparing a glycosylated hemoglobin calibrator and a preparation method thereof. The kit comprises: a cell stock solution comprising: phosphate buffer, sodium chloride; a saccharification liquid comprising: phosphate buffer, sodium chloride and glucose; a hemolytic agent comprising: phosphate buffer solution, sodium chloride and first surfactant; a lyoprotectant comprising: phosphate buffer solution, sodium chloride, antioxidant and second surfactant. Through the mode, the method can avoid the problem that a diabetic blood sample which is not easy to obtain is used for preparing the glycosylated hemoglobin calibrator in the prior art, so that the limitation of preparation raw materials is reduced to a certain extent, and the preparation cost is reduced.
Description
Technical Field
The application relates to the technical field of biology, in particular to a kit for preparing a glycosylated hemoglobin calibrator and a preparation method thereof.
Background
Glycated hemoglobin HbA1c is a diabetes diagnosis standard recommended by the world health organization (2011), is a 'gold standard' for controlling blood sugar of diabetes, and is an effective index for evaluating diabetic microvascular and macrovascular complications. Fresh diabetic blood samples are often selected as glycated hemoglobin calibrators, but there are many limitations in acquisition and use, and it is therefore highly desirable to prepare a calibrator that is stable for use in everyday tasks.
Disclosure of Invention
The present application is directed to solving, at least in part, one of the technical problems in the related art.
In a first aspect of the present application, there is provided a kit for preparing a glycated hemoglobin calibrator, the kit comprising: a cell storage fluid comprising: phosphate buffer, sodium chloride; a saccharification liquid comprising: phosphate buffer, sodium chloride and glucose; a hemolytic agent comprising: phosphate buffer solution, sodium chloride and first surfactant; a lyoprotectant comprising: phosphate buffer solution, sodium chloride, antioxidant and second surfactant.
In a second aspect of the present application, the present application provides a method for preparing a glycated hemoglobin calibrator, which is based on the kit, comprising the steps of: weighing cell storage liquid according to a formula, and cleaning erythrocytes by using the cell storage liquid; weighing a saccharification solution according to the formula, and performing glycosylation treatment on the cleaned red blood cells by adopting the saccharification solution until the concentration of the glycosylated hemoglobin in the red blood cells reaches a preset concentration; weighing cell stock solution according to the formula, washing the glycosylated red blood cells by using the cell stock solution, and performing deglycosylation; weighing a hemolytic agent according to a formula, and treating erythrocytes by using the hemolytic agent to obtain glycosylated hemoglobin; weighing a freeze-drying protective agent according to the formula, and carrying out freeze-drying treatment on the glycosylated hemoglobin by adopting the freeze-drying protective agent to obtain the glycosylated hemoglobin calibrator.
The technical scheme provided by the embodiment of the application can bring the following beneficial effects:
compared with the prior art, the kit comprises: the cell storage solution, the saccharified solution, the hemolytic agent and the freeze-drying protective agent can perform an in-vitro glycosylation reaction with red blood cells in a healthy and normal human blood sample, so that glycosylated hemoglobin calibrators with different concentrations are prepared, the problem that in the prior art, a diabetic blood sample which is not easy to obtain is used for preparing the glycosylated hemoglobin calibrators is avoided, the limitation of preparation raw materials is reduced to a certain extent, and the preparation cost is reduced.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts. Wherein:
FIG. 1 is a schematic flow diagram illustrating a method of preparing a glycated hemoglobin calibrator according to the present application;
FIG. 2 shows a glycated hemoglobin measurement system (assessment system) calibrated using the glycated hemoglobin calibrator of example 1 of the present application, and a correlation test when different concentrations of glycated hemoglobin samples are measured using a Bio-Rad D-100 glycated hemoglobin analysis system (reference system).
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The application provides a kit for preparing a glycosylated hemoglobin calibrator, which comprises a cell storage solution, a glycosylated solution, a hemolytic agent and a lyoprotectant which are independently packaged.
Wherein, cell stock solution includes: phosphate buffer, sodium chloride.
The saccharification liquid comprises: phosphate buffer, sodium chloride and glucose.
The hemolytic agent comprises: phosphate buffer, sodium chloride and a first surfactant.
Freeze-drying protective agent: the method comprises the following steps: phosphate buffer solution, sodium chloride, antioxidant and second surfactant.
Specifically, the glycated liquid is used to produce glycated hemoglobin by a glycosylation reaction with hemoglobin in red blood cells. Hemolytic agents are used to treat red blood cells to release glycated hemoglobin. Lyoprotectants are used to improve the transport and shelf life of glycated hemoglobin at room temperature.
Phosphate buffer was used to adjust the pH of the solution. Sodium chloride is used to adjust the ionic strength of the solution to avoid causing changes in the osmotic pressure of the cells. The glucose is used for glycosylation reaction with hemoglobin in red blood cells to obtain glycosylated hemoglobin.
The first surfactant can be an anionic surfactant or a nonionic surfactant, and in other embodiments, the first surfactant can also be other types of surfactants. Wherein the anionic surfactant has functions of breaking cell and dissolving erythrocyte, and can combine with hemoglobin to form stable complex, and the anionic surfactant can be selected from one of alkyl sulfonate, alkylbenzene sulfonate, and alkyl sulfate. The nonionic surfactant can participate in the dissolution of red blood cells, accelerate the formation of a hemoglobin compound, increase the dissolving capacity of the anionic surfactant, prevent the anionic surfactant from being separated out at low temperature and enhance the stability of the hemoglobin compound. The nonionic surfactant can be polyoxyethylene ether, specifically polyethylene glycol octyl phenyl ether Triton X-100.
The antioxidant is used for preventing oxidation reaction during the preservation process of the glycosylated hemoglobin. The antioxidant is selected from at least one of vitamin C and its derivatives, vitamin E and its derivatives, butyl hydroxy anisole (BIA), tea polyphenols, sesamol or guaiac resin.
The second surfactant can be mannitol and polyethylene glycol, wherein mannitol can be used as filler, also contains a plurality of hydroxyl groups, and can replace water to form hydrogen bonds with hydrophilic groups of the glycosylated hemoglobin, so that the spatial structure of the glycosylated hemoglobin is protected, and coagulation is avoided. The polyethylene glycol can provide a frame structure, improve the toughness after freeze-drying and is beneficial to maintaining the shape after freeze-drying.
Compared with the prior art, the kit comprises: the cell storage solution, the saccharified solution, the hemolytic agent and the freeze-drying protective agent can perform an in-vitro glycosylation reaction with red blood cells in a healthy and normal human blood sample, so that glycosylated hemoglobin calibrators with different concentrations are prepared, the problem that in the prior art, a diabetic blood sample which is not easy to obtain is used for preparing the glycosylated hemoglobin calibrators is avoided, the limitation of preparation raw materials is reduced to a certain extent, and the preparation cost is reduced.
In one embodiment, at least one of the saccharification liquid, the cell storage liquid, the hemolytic agent, and the lyoprotectant further comprises: and (4) a preservative.
Optionally, the preservative is at least one of sodium benzoate, sodium azide, potassium sorbate, 2-methyl-4-isothiazolin-3-one, proclin-300.
In one embodiment, the first surfactant includes at least: triton X-100 and dodecyl polyethylene glycol ether (aka benzyl luster 35). The second surfactant includes at least: polyethylene glycol, mannitol. The antioxidant comprises at least: d-sodium erythorbate.
In one embodiment, a cell stock solution comprises: 50-70 mmol/L of disodium hydrogen phosphate, 50-70 mmol/L of sodium dihydrogen phosphate, 0.05-0.1 percent of sodium chloride (mass percent concentration), 3000.03-0.05 percent of Proclin (mass percent concentration) and the balance of purified water, wherein the pH value of the cell storage solution is 7.2-7.4.
Preferably, the cell stock solution comprises: 50mmol/L disodium hydrogen phosphate, 50mmol/L sodium dihydrogen phosphate, 0.09% (mass percent concentration) sodium chloride, 0.03% (mass percent concentration) Proclin, and the balance of purified water, wherein the pH value of the cell stock solution is 7.3.
In one embodiment, the saccharification liquid comprises: 50-70 mmol/L of disodium hydrogen phosphate, 50-70 mmol/L of sodium dihydrogen phosphate, 0.05-0.1 percent of sodium chloride (mass percentage concentration), 1-2 percent of glucose (mass percentage concentration), 0.03-0.05 percent of Proclin-300 (mass percentage concentration) and the balance of purified water, wherein the pH value of the saccharification liquid is 7.2-7.4.
Preferably, the saccharification liquid comprises: 50mmol/L disodium hydrogen phosphate, 50mmol/L sodium dihydrogen phosphate, 0.09% (mass percent concentration) sodium chloride, 1% (mass percent concentration) glucose, 0.03% (mass percent concentration) Proclin and the balance of purified water, wherein the pH value of the saccharification liquid is 7.3.
In one embodiment, the hemolytic agent comprises: 30-50 mmol/L disodium hydrogen phosphate, 30-50 mmol/L sodium dihydrogen phosphate, 0.01-0.05% (mass percent concentration) sodium chloride, 3000.03-0.05% (mass percent concentration) Proclin, 0.05-0.1% (mass percent concentration) Triton X-100, 0.05-0.1% (mass percent concentration) dodecyl polyglycol ether and the balance of purified water, wherein the pH value of the hemolytic agent is 7.2-7.4.
Preferably, the hemolytic agent comprises: 30mmol/L disodium hydrogen phosphate, 30mmol/L sodium dihydrogen phosphate, 0.01% (mass percent concentration) sodium chloride, 0.03% (mass percent concentration) Proclin, 100.05% (mass percent concentration) Triton X, 0.05% (mass percent concentration) dodecyl polyglycol ether and the balance of purified water, wherein the pH value of the hemolytic agent is 7.3.
In one embodiment, the lyoprotectant includes: 50-70 mmol/L of disodium hydrogen phosphate, 50-70 mmol/L of sodium dihydrogen phosphate, 0.05-0.1 percent of sodium chloride (mass percent concentration), 3000.03-0.05 percent of Proclin (mass percent concentration), 1-2 percent of mannitol (mass percent concentration), 1-2 percent of polyethylene glycol (6000), 0.05-0.1 percent of D-sodium erythorbate (mass percent concentration) and the balance of purified water.
Preferably, the lyoprotectant comprises: 50mmol/L disodium hydrogen phosphate, 50mmol/L sodium dihydrogen phosphate, 0.09% (mass percent concentration) sodium chloride, 0.03% (mass percent concentration) Proclin-300, 2% (mass percent concentration) mannitol, 1% (mass percent concentration) polyethylene glycol (6000), 0.05% (mass percent concentration) D-sodium erythorbate and the balance of purified water.
The application also provides a preparation method of the glycosylated hemoglobin calibrator, the preparation method is based on the kit, and the preparation method comprises the following steps:
s10: weighing the cell storage solution according to the formula, and cleaning the red blood cells by adopting the cell storage solution.
In the above step 10, the volume ratio of the cell stock solution to the red blood cells per washing is 3.
Specifically, erythrocytes were washed 3 times to remove impurities and obtain erythrocytes with higher purity, by centrifuging at 3000rpm for 5min at 4 ℃ according to a volume ratio of the cell stock solution to erythrocytes of 4.
S20: and (3) weighing the saccharification liquid according to the formula, and performing glycosylation treatment on the cleaned red blood cells by adopting the saccharification liquid until the concentration of the glycosylated hemoglobin in the red blood cells reaches a preset concentration.
In the step 20, the volume ratio of the glycated liquid to the red blood cells is 1, the temperature of the glycosylation treatment is 36 to 38 ℃, the reaction time of the glycosylation treatment is 24 to 72 hours, and a DM glycated hemoglobin analysis system is adopted to monitor the glycated hemoglobin concentration in the red blood cells in real time.
Specifically, a saccharification liquid is added to the washed erythrocytes to perform in vitro glycosylation treatment, the volume ratio of the saccharification liquid to the erythrocytes is 1. During the glycosylation process, the concentration of glycated hemoglobin in erythrocytes can be monitored in real time by using a DM glycated hemoglobin analysis system (HPLC method) at 12 hours, 24 hours, 36 hours, 42 hours, and 48 hours, respectively, and the process proceeds to step S30 until the concentration of glycated hemoglobin formed reaches a predetermined concentration. Wherein, the preset concentration may be: the mass percentage concentration of the glycosylated hemoglobin is 9 to 11 percent.
S30: the cell stock solution is weighed according to the formula, and the red blood cells are washed by using the cell stock solution to remove glucose and prevent further glycosylation of hemoglobin.
In the step 30, the volume ratio of the cell stock solution to the red blood cells is 3.
Specifically, the erythrocytes were washed 3 times to remove glucose and prevent further glycosylation of hemoglobin according to a cell stock to erythrocyte volume ratio of 4.
S40: weighing hemolytic agent according to the formula, and treating red blood cells by adopting hemolytic agent to obtain glycosylated hemoglobin.
In the above step 40, the volume ratio of the hemolytic agent to the red blood cells is 1.
Specifically, adding hemolytic agent with the same volume as that of red blood cells, mixing uniformly, standing for 5-10 min, centrifuging at 4 ℃ for 5min at 3000rpm, and retaining supernatant containing glycosylated hemoglobin.
S50: weighing a freeze-drying protective agent according to the formula, and carrying out freeze-drying treatment on the glycosylated hemoglobin by adopting the freeze-drying protective agent to obtain the glycosylated hemoglobin calibrator.
Specifically, a lyoprotectant is added to the supernatant obtained in step S40, and then a lyophilization process is performed, and after the lyophilization process is completed, a glycated hemoglobin calibrator can be obtained.
Wherein, the freeze-drying treatment comprises the following steps:
s501: and (6) pre-freezing. In the step, the pre-freezing time is set to be 60-90 min, the temperature of the slab layer is-40 ℃, and the duration time is 120-180 min.
S502: and (5) primary drying. In the step, the primary drying time is set to be 20-40 min, the temperature of the plate layer is-25 ℃, the vacuum degree is 0.15mBar, and the duration time is 720-900 min.
S503: and (4) resolving and drying. In the step, the analysis drying time is set to be 300min, the temperature of the plate layer is-30 ℃, the vacuum degree is 0.15mBar, and the duration time is 300-360 min.
The application also provides a preparation method of the glycosylated hemoglobin calibrator, the preparation method is based on the kit, and the preparation method comprises the following steps:
s60: a blood sample is provided.
Wherein, in the blood sample, the red blood cell count is 3.5X 10 12 ～5.5×10 12 The concentration of the hemoglobin is 120-160 g/L, and the mass percentage concentration of the glycosylated hemoglobin is 4.5-5.5%.
S70: and (4) carrying out centrifugal treatment on the blood sample to obtain the red blood cells.
Specifically, after the biological safety of the blood sample is qualified, the blood sample is centrifuged at 3000rpm for 5min at 4 ℃, and the lowest red precipitate is retained to obtain red blood cells.
The present application will be described in detail with reference to specific examples. The following examples will aid those skilled in the art in further understanding the present application, but are not intended to limit the present application in any way. It should be noted that numerous variations and modifications could be made by those skilled in the art without departing from the spirit of the application. All falling within the scope of protection of the present application.
Experimental example 1
The method for preparing the glycosylated hemoglobin calibrator with low concentration level comprises the following steps:
step 101: blood samples were collected (red blood cell count in blood samples was 3.5X 10) 12 ～5.5×10 12 The hemoglobin concentration is 120-160 g/L, the mass percent concentration of the glycosylated hemoglobin is 4.5-5.5 percent), after the biological safety of the blood sample is qualified, the blood sample is centrifuged for 5min at 3000rpm at 4 ℃, and the lowest red precipitate is reserved to obtain the red blood cells.
Step 102: and (3) centrifuging at 4 ℃ for 5min by adopting 3000rpm according to the volume ratio of the cell stock solution to the red blood cells being 4.
Step 103: adding hemolytic agent with the same volume as red blood cells, uniformly mixing, standing for 5-10 min, determining the concentration of the glycosylated hemoglobin by adopting a DM glycosylated hemoglobin analysis system (HPLC method), wherein the mass percent concentration of the glycosylated hemoglobin is in a range of 4.5-5.5%, centrifuging for 5min at 3000rpm at 4 ℃, and reserving supernate containing the glycosylated hemoglobin.
Step 104: and adding a freeze-drying protective agent into the supernatant, carrying out freeze-drying treatment, and obtaining the glycosylated hemoglobin calibrator with a low concentration level after freeze-drying.
TABLE 1 Components and concentrations of cell stock solution, saccharification solution, hemolytic agent, and lyoprotectant used in example 1
Note: "/" indicates the absence of this component.
The lyophilization process parameters in step 104 are shown in table 2.
TABLE 2 Freeze drying Process parameters
Example 2
The method for preparing the glycosylated hemoglobin calibrator of the high-concentration HbA1c calibrator comprises the following steps of:
step 201: blood samples were collected (red blood cell count in blood samples was 3.5X 10) 12 ～5.5×10 12 The hemoglobin concentration is 120-160 g/L, the mass percent concentration of the glycosylated hemoglobin is 4.5-5.5 percent), after the biological safety of the blood sample is qualified, the blood sample is centrifuged for 5min at 3000rpm at 4 ℃, and the lowest red precipitate is reserved to obtain the red blood cells.
Step 202: and (3) centrifuging at 3000rpm for 5min at 4 ℃ according to the volume ratio of the cell stock solution to the red blood cells of 4, and washing the red blood cells for 3 times to remove impurities so as to obtain the red blood cells with higher purity.
Step 203: adding a saccharification liquid into the washed red blood cells to perform in-vitro glycosylation treatment, wherein the volume ratio of the saccharification liquid to the red blood cells is 1.
Step 204: the red blood cells were washed 3 times to remove glucose and prevent further glycosylation of hemoglobin according to a cell stock to red blood cell volume ratio of 4.
Step 205: adding hemolytic agent with the same volume with erythrocyte, mixing uniformly, standing for 5-10 min, centrifuging at 4 deg.C and 3000rpm for 5min, and retaining supernatant containing glycosylated hemoglobin.
Step 206: and (3) adding a freeze-drying protective agent into the supernatant obtained in the step (205), and then carrying out freeze-drying treatment, so as to obtain the glycosylated hemoglobin calibrator with a high concentration level.
TABLE 3 Components and concentrations of cell stock solution, saccharification solution, hemolytic agent and lyoprotectant used in example 2
Note: "/" indicates the absence of this component.
The lyophilization process parameters in step 206 are shown in table 4.
TABLE 4 Freeze-drying Process parameters
Example 3
Performance parameter testing of Low and high concentration level calibrators
1. Accuracy of
After calibration using the normal value national standard and the abnormal value national standard, the low concentration level calibrator and the high concentration level calibrator were measured on the glycated hemoglobin measurement system, and the accuracy test results are shown in table 5.
TABLE 5 accuracy test results
As is clear from the results of Table 5, the glycated hemoglobin calibrators prepared in examples 1 and 2 at the low concentration level and the high concentration level, respectively, had relative deviations from the indicated values within. + -. 10%, and thus satisfied the accuracy requirements.
2. Uniformity of
Requires a Coefficient of Variation (CV) in uniformity between bottles Bottle room ) Should be not more than 3.0%, and coefficient of variation of uniformity (CV) in CV bottle In the bottle ) Should not be greater than 1.5%. The results of the homogeneity test for the glycated hemoglobin calibrator at the low concentration level are shown in Table 6, and the results of the homogeneity test for the glycated hemoglobin calibrator at the high concentration level are shown in Table 7.
TABLE 6 results of homogeneity test of glycated hemoglobin calibrator at Low concentration levels
TABLE 7 results of homogeneity test of glycated hemoglobin calibrator at high concentration levels
As is clear from the results in tables 6 and 7, the glycated hemoglobin standards at the low concentration level and the high concentration level obtained in examples 1 and 2, respectively, satisfy the requirement of uniformity.
3. Stability of
Standing at 37 deg.C under accelerated condition for 16 days; after redissolving, the mixture is placed at 2-8 ℃ for 40 days, and the relative deviation of the obtained test result and the indicated value is required to be within the range of +/-10%. The results of the accelerated stability test of the glycated hemoglobin calibrator at the low concentration level are shown in Table 8, and the results of the accelerated stability test of the glycated hemoglobin calibrator at the high concentration level are shown in Table 9. The results of the open bottle stability test after reconstitution of the glycated hemoglobin calibrator at a low concentration level are shown in Table 10, and the results of the open bottle stability test after reconstitution of the glycated hemoglobin calibrator at a high concentration level are shown in Table 11.
TABLE 8 accelerated stability test results for glycated hemoglobin calibrator at Low concentration levels
TABLE 9 accelerated stability test results for glycated hemoglobin calibrator at high concentration levels
TABLE 10 post reconstitution decap stability test results for glycated hemoglobin calibrators at low concentration levels
TABLE 11 results of post reconstitution decap stability test of glycated hemoglobin calibrator at high concentration levels
From the results of tables 8 to 11, it is understood that the glycated hemoglobin calibrators prepared in examples 1 and 2 both at a low concentration level and at a high concentration level satisfy the stability requirements.
4. Correlation
When a glycated hemoglobin measurement system (assessment system) calibrated by using the glycated hemoglobin calibrator of example 1 of the present application and a Bio-Rad D-100 glycated hemoglobin analysis system (reference system) are used to measure glycated hemoglobin samples with different concentrations, it is required that the measurement results are consistent, i.e., the slope k =1.00 ± 0.10 and the correlation coefficient r is not less than 0.975. The results of the correlation tests are shown in table 12 and figure 2 of the specification.
TABLE 12 correlation test results
As is clear from the results in Table 12 and FIG. 2, the glycated hemoglobin calibrators prepared in examples 1 and 2 both at a low concentration level and a high concentration level satisfy the correlation requirements.
Compared with the prior art, the kit comprises: the cell storage solution, the saccharified solution, the hemolytic agent and the freeze-drying protective agent can perform an in-vitro glycosylation reaction with red blood cells in a healthy and normal human blood sample, so that glycosylated hemoglobin calibrators with different concentrations are prepared, the problem that in the prior art, a diabetic blood sample which is not easy to obtain is used for preparing the glycosylated hemoglobin calibrators is avoided, the limitation of preparation raw materials is reduced to a certain extent, and the preparation cost is reduced.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (14)
1. A kit for preparing a glycated hemoglobin calibrator, comprising:
a cell storage fluid comprising: phosphate buffer, sodium chloride;
a saccharification fluid comprising: phosphate buffer, sodium chloride and glucose;
a hemolytic agent comprising: phosphate buffer solution, sodium chloride and first surfactant;
a lyoprotectant comprising: phosphate buffer solution, sodium chloride, antioxidant and second surfactant.
2. The kit of claim 1, wherein at least one of the saccharification fluid, the cell stock solution, the hemolytic agent, and the lyoprotectant further comprises: and (4) a preservative.
3. The kit according to claim 2,
the preservative is at least one of sodium benzoate, sodium azide, potassium sorbate, 2-methyl-4-isothiazoline-3-ketone and Proclin-300.
4. The kit according to claim 1,
the first surfactant includes at least: triton X-100 and dodecyl polyglycol ether;
the second surfactant includes at least: polyethylene glycol, mannitol;
the antioxidant at least comprises: d-sodium erythorbate.
5. The kit according to claim 2,
the cell stock solution comprises: 50-70 mmol/L of disodium hydrogen phosphate, 50-70 mmol/L of sodium dihydrogen phosphate, 0.05-0.1 percent of sodium chloride (mass percent concentration), 0.03-0.05 percent of Proclin-300 (mass percent concentration) and the balance of purified water, wherein the pH value of the cell stock solution is 7.2-7.4.
6. The kit according to claim 2,
the saccharification liquid comprises: 50-70 mmol/L of disodium hydrogen phosphate, 50-70 mmol/L of sodium dihydrogen phosphate, 0.05-0.1 percent of sodium chloride (mass percentage concentration), 1-2 percent of glucose (mass percentage concentration), 0.03-0.05 percent of Proclin-300 (mass percentage concentration) and the balance of purified water, wherein the pH value of the saccharification liquid is 7.2-7.4.
7. The kit according to claim 2,
the hemolytic agent comprises: 30-50 mmol/L disodium hydrogen phosphate, 30-50 mmol/L sodium dihydrogen phosphate, 0.01-0.05% (mass percent concentration) sodium chloride, 0.03-0.05% (mass percent concentration) Proclin-300, 0.05-0.1% (mass percent concentration) Triton X-100, 0.05-0.1% (mass percent concentration) dodecyl polyglycol ether, and the balance of purified water, wherein the pH value of the hemolytic agent is 7.2-7.4.
8. The kit according to claim 2,
the lyoprotectant includes: 50-70 mmol/L of disodium hydrogen phosphate, 50-70 mmol/L of sodium dihydrogen phosphate, 0.05-0.1 percent (mass percentage concentration) of sodium chloride, 0.03-0.05 percent (mass percentage concentration) of Proclin-300, 1-2 percent (mass percentage concentration) of mannitol, 1-2 percent (mass percentage concentration) of polyethylene glycol (6000), 0.05-0.1 percent (mass percentage concentration) of D-sodium erythorbate and the balance of purified water.
9. A method for preparing a glycated hemoglobin calibrator, the method being based on the kit according to any one of claims 1 to 8, the method comprising the steps of:
weighing cell storage liquid according to a formula, and cleaning erythrocytes by using the cell storage liquid;
weighing a saccharification liquid according to a formula, and performing glycosylation treatment on the cleaned red blood cells by adopting the saccharification liquid until the concentration of the glycosylated hemoglobin in the red blood cells reaches a preset concentration;
weighing the cell stock solution according to a formula, and washing the glycosylated erythrocytes by using the cell stock solution for deglycosylation;
weighing a hemolytic agent according to a formula, and treating the red blood cells by using the hemolytic agent to obtain glycosylated hemoglobin;
weighing a freeze-drying protective agent according to a formula, and carrying out freeze-drying treatment on the glycosylated hemoglobin by adopting the freeze-drying protective agent to obtain a glycosylated hemoglobin calibrator.
10. The method of claim 9, wherein prior to the step of weighing the cell stock according to the formula and washing the red blood cells with the cell stock, the method further comprises:
providing a blood sample;
centrifuging the blood sample to obtain the red blood cells;
wherein in the blood sample, the red blood cell count is 3.5X 10 12 ～5.5×10 12 The concentration of the hemoglobin is 120-160 g/L, and the mass percentage concentration of the glycosylated hemoglobin is 4.5-5.5%.
11. The method of claim 9, wherein in the step of weighing the cell stock solution according to the formula and washing the red blood cells with the cell stock solution, the volume ratio of the cell stock solution to the red blood cells is 3.
12. The method according to claim 9, wherein in the step of weighing a saccharification liquid according to a formula and performing glycosylation treatment on the washed red blood cells by using the saccharification liquid until the concentration of the glycated hemoglobin in the red blood cells reaches a preset concentration, the volume ratio of the saccharification liquid to the red blood cells is 1.
13. The method of claim 9, wherein in the step of weighing the cell stock solution according to the formula and washing the red blood cells with the cell stock solution to remove glucose and prevent further glycosylation of hemoglobin, the volume ratio of the cell stock solution to the red blood cells is 3 to 5.
14. The method according to claim 9, wherein in the step of weighing the lyoprotectant according to the formulation and performing lyophilization treatment on the glycated hemoglobin by using the lyoprotectant to obtain the glycated hemoglobin calibrator, the volume ratio of the hemolytic agent to the red blood cells is 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116625777A (en) * | 2023-07-21 | 2023-08-22 | 山东新华医疗器械股份有限公司 | Low-level freeze-dried glycosylated hemoglobin control and preparation method thereof |
| CN116625777B (en) * | 2023-07-21 | 2023-10-13 | 山东新华医疗器械股份有限公司 | Low-level freeze-dried glycosylated hemoglobin control and preparation method thereof |
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