CN105738634A - Method for preparing glycosylated hemoglobin quality control material - Google Patents
Method for preparing glycosylated hemoglobin quality control material Download PDFInfo
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Abstract
The invention discloses a method for preparing a glycosylated hemoglobin quality control material. The method comprises the steps of conducting centrifugal treatment on a blood sample, so that red cells are obtained; washing the red cells with cell washing liquid, so that red cell suspension is formed; adding a reaction solution to the washed red cells for glycosylation; when the concentration of glycosylated hemoglobin reaches the required quality control material concentration in the glycosylation process, washing the red cell suspension obtained after glycosylation treatment with cell washing liquid, so that deglycosylation is conducted; adding a stabilizing agent and a freeze-drying protective agent to the red cell suspension obtained after deglycosylation, so that the liquid or freeze-dried glycosylated hemoglobin quality control material is obtained. By means of the method, the glycosylated hemoglobin quality control material high in stability can be obtained.
Description
Technical field
The present invention relates to technical field of medical examination, the preparation method particularly relating to a kind of glycolated hemoglobin Quality Control thing.
Background technology
Treating diabetes is it is crucial that the control of blood sugar level is with stable, and glycolated hemoglobin is the glycosylated product of hemoglobin, and it can reflect the average blood glucose levels in past 8～12 weeks of patient, and not by the impact of parameters of short term glycemic fluctuation of concentration.The Accurate Determining needs one of this glycolated hemoglobin are stablized Quality Control product and are carried out the quality of monitoring instrument state and reagent.And existing Quality Control product stability is poor, so that the mensuration of glycolated hemoglobin exists deviation.
Summary of the invention
Present invention is primarily targeted at the preparation method that a kind of glycolated hemoglobin Quality Control thing is provided, it is intended to obtain stability height glycolated hemoglobin Quality Control thing.
For achieving the above object, the preparation method that the present invention provides a kind of glycolated hemoglobin Quality Control thing, comprise the following steps:
S1, is centrifuged blood sample processing, it is thus achieved that erythrocyte；
S2, utilizes cell washing solution that described erythrocyte is washed, and forms red cell suspension；
S3, carries out glycosylation in erythrocyte addition reactant liquor after washing；
S4, when the concentration of glycolated hemoglobin reaches required Quality Control substrate concentration in glycosylation process, utilizes cell washing solution that the red cell suspension of described step S3 is washed, carries out deglycosylation；
S5, adds stabilizer, freeze drying protectant in deglycosylated red cell suspension, makes the glycolated hemoglobin Quality Control thing of liquid or lyophilizing.
Preferably, also include before described step S1:
S0, adds antioxidant in blood sample.
Preferably, the composition of described cell detergent is:
Polyethylene glycol: 0～3%；
Disodium: 0～3%；
Magnesium gluconate: 0-1%；
Na2HPO4:0-2%；
Non-glucose hexahydroxylic alcohols hexose: 0-8%；
Methyl parahydroxybenzoate: 0-0.2%；
Inosine: 0-0.5%；
Neomycin: 0-0.2%；
Chloromycetin: 0-0.2%；
(NaOH): 0-0.5%；
(KCl): 0-1.5%；
Osmolarity: 250-300mOsm/L；
Above-mentioned solvent is deionized water.
Preferably, described non-glucose hexahydroxylic alcohols hexose is mannose.
Preferably, the described composition containing mannose or the reactant liquor of glucose is:
Polyethylene glycol (200-50000): 0-3%；
Disodium: 0-3%；
Magnesium gluconate: 0-1%；
Na2HPO4:0-2%；
Mannose or glucose: 0-8%；
Methyl parahydroxybenzoate: 0-0.2%；
Inosine: 0-0.6%；
Neomycin: 0-0.2%；
Chloromycetin: 0-0.2%；
(NaOH): 0-0.05%；
(KCl): 0-1.5%；
NaF:0～0.5%；
Ph:6.0～8.0；
Osmolarity: 250-300mOsm/L；
Above-mentioned solvent is deionized water.
Preferably, described reactant liquor is additionally added bovine serum albumin, and the mass concentration that described bovine serum albumin is in reactant liquor has been 1～6%.
Preferably, reaction temperature when carrying out glycosylation in described step S3 is 18～23 DEG C, and the response time is 4～8 days.
Preferably, in described blood sample, erythrocytic concentration is: 3.5*1012/L-5.0*1012/L, and the concentration of hemoglobin is: 9g/L-12g/L.
Preferably, in described step S4, the red cell suspension after glycosylation is added cell washing solution, and carries out being centrifuged off precipitation, leave and take supernatant.
Preferably, described red cell suspension is minimum for 1:3 with the ratio of cell washing solution.
The erythrocyte of embodiment of the present invention employing normal person carries out external glycosylation and carries out the preparation of glycolated hemoglobin Quality Control thing, expands the source of raw material.It addition, the embodiment of the present invention utilizes non-glucose hexahydroxylic alcohols hexose as external glycosylated saccharide, shorten the external glycosylated time.And, the embodiment of the present invention also utilizes cell washing solution to carry out deglycosylation process so that the concentration of glycolated hemoglobin unstable in the glycolated hemoglobin Quality Control thing after preparation reduces, thus ensure that the stability of glycolated hemoglobin Quality Control thing.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet of the preparation method first embodiment of glycolated hemoglobin Quality Control thing of the present invention；
Fig. 2 is that the erythrocytic hemoglobin of the present invention carries out glycosylated process；
Fig. 3 is the schematic flow sheet of preparation method the second embodiment being glycolated hemoglobin Quality Control thing of the present invention.
The realization of the object of the invention, functional characteristics and advantage will in conjunction with the embodiments, are described further with reference to accompanying drawing.
Detailed description of the invention
Should be appreciated that specific embodiment described herein is only in order to explain the present invention, is not intended to limit the present invention.
The preparation method that the present invention provides a kind of glycolated hemoglobin Quality Control thing.As it is shown in figure 1, this method processed can comprise the following steps that
S1, is centrifuged blood sample processing, it is thus achieved that erythrocyte；
Above-mentioned blood sample may be from the healthy population not having diabetes, naturally it is also possible to for other sources.After collecting blood sample, can be centrifuged this blood sample processing, it is thus achieved that erythrocyte.The concentration of the blood rbc adopted is preferably the same with the concentration in the blood of normal person.Specifically, in blood sample, erythrocytic concentration is: 3.5*1012/L-5.0*1012/L, and the concentration of hemoglobin is: 9g/L-12g/L.
S2, utilizes cell washing solution that described erythrocyte is washed, and forms red cell suspension；
Erythrocyte adds cell washing solution, it is thus achieved that erythrocyte mixture, i.e. red cell suspension.The ratio of this erythrocyte and cell washing solution can be arranged as the case may be and flexibly, and washing times can also be arranged flexibly, and in the present embodiment, this washing times is 3 times.Composition and the concentration of above-mentioned cell washing solution are as shown in table 1 below:
The composition of table 1. cell washing solution and concentration
| Cell washing solution composition | Concentration |
| Polyethylene glycol | 0-3% |
| Ethylenediaminetetraacetic acid (EDTA) | 0-3% |
| Magnesium gluconate | 0-1% |
| Na2HPO4 | 0-2% |
| Mannose | 0-8% |
| Methyl parahydroxybenzoate | 0-0.2% |
| Inosine | 0-0.5% |
| Neomycin | 0-0.2% |
| Chloromycetin | 0-0.2% |
| (NaOH) | 0-0.5% |
| (KCl) | 0-1.5% |
| Osmolarity | 250-300mOsm/L |
Above-mentioned solvent is deionized water.In upper table 1, mannose can also replace with other non-glucose hexahydroxylic alcohols hexose or glucose, in the present embodiment, it is preferred to mannose.In the embodiment of the present invention, the concentration of this mannose is as far as possible low, even for 0%, in order to avoid erythrocytic hemoglobin and mannose carry out glycosylation.Above-mentioned Na2HPO4 uses as phosphate buffer, naturally it is also possible to replaced by other buffer, for instance Tri(Hydroxymethyl) Amino Methane Hydrochloride buffer, acetic acid-sodium acetate buffer solution etc..
Preferably, the concentration example of the composition of above-mentioned cell washing solution is as shown in table 2 below:
| Cell washing solution composition | Concentration |
| Polyethylene glycol | 0.7% |
| EDTA | 0.7% |
| Magnesium gluconate | 0.39% |
| Na2HPO4 | 0.27% |
| Mannose | 0% |
| Methyl parahydroxybenzoate | 0.04% |
| Inosine | 0.025% |
| Neomycin | 0.04% |
| Chloromycetin | 0.015% |
| (NaOH) | 0.08% |
| (KCl) | 0.632% |
| PH | 7.0 |
| Osmolarity | 300mOsm/L |
S3, adds in reactant liquor in red cell suspension after washing and carries out glycosylation；
Composition and the concentration of above-mentioned reactant liquor are as shown in table 3 below:
After red cell suspension mixes with the reactant liquor containing mannose, hemoglobin in erythrocyte can carry out glycosylation, and the glycosylation stage can produce the glycolated hemoglobin (Labile-A1c of instability, and stable glycolated hemoglobin (Stable-A1c L-A1C), S-A1c), as shown in Figure 2.Wherein, unstable glycolated hemoglobin is Rapid reversible, in the mannose of no or low concentration, will be broken down into hemoglobin and mannose.Stable glycolated hemoglobin is irreversible.Due in glycosylation process, the relative ratios that converts of stable glycolated hemoglobin depends on the amount of mannose in reactant liquor, therefore by arranging the amount of mannose in reactant liquor, may decide that glycolated hemoglobin concentration in erythrocyte, thus preparing a series of different glycolated hemoglobin Quality Control thing, for various applications.It is preferred that stable glycolated hemoglobin concentration in erythrocyte accounts for the concentration of gross weight 6%-12% or higher.Generally, the normal concentration range of the glycolated hemoglobin S-A1c of normal person is to account for the 6% of erythrocyte gross weight or following.
In order to obtain the glycolated hemoglobin Quality Control thing of desired concn, needing the mannose containing q.s so that glycolated hemoglobin is to expection concentration in above-mentioned reactant liquor, mannose concentration limit in reactant liquor is can with the concentration of rational speed glycolated hemoglobin.In the embodiment of the present invention, mannose mass percent in reactant liquor is at 2%-8%.And, ambient temperature during glycosylation is more high, and the response time required for reaching the glycolated hemoglobin Quality Control thing of desired concn is more short.In process of the test, it has been found that temperature is too high, although the response time can be reduced, but the less stable of the glycolated hemoglobin Quality Control thing after preparation.Therefore, in the embodiment of the present invention, erythrocytic hemoglobin carries out reaction temperature when glycosylation processes is 18～23 DEG C, and the response time is 4～8 days.
Further, above-mentioned reactant liquor also can add bovine serum albumin, and the mass concentration that described bovine serum albumin is in reactant liquor is 1～6%.Reactant liquor adds bovine serum albumin, it is therefore an objective to anticoagulation and anti-hemolysis.In reactant liquor, the concentration limit of bovine serum albumin is the minimum bovine serum albumin concentration that can prevent haemolysis and cohesion, and the upper limit of bovine serum albumin concentration is that concentration increase does not further reduce blood coagulation, haemolysis.In the embodiment of the present invention, this bovine serum albumin concentration in reactant liquor is preferably between 1%-6%.
S4, when the concentration of glycolated hemoglobin reaches required Quality Control substrate concentration in glycosylation process, utilizes cell washing solution that the red cell suspension of described step S3 is washed, carries out deglycosylation；
The unstable glycolated hemoglobin (L-A1C) of higher concentration would generally be comprised, although L-A1c there is also in the blood of diabetics, but concentration is relatively low after hemoglobin glycosylation in erythrocyte.Accordingly, it would be desirable to the erythrocyte deglycosylation after glycosylation so that the concentration of unstable glycolated hemoglobin reduces.In glycosylation process, the concentration of stable glycolated hemoglobin will be monitored in real time, high performance liquid chromatography (HPLC) can be adopted, method that immunoassay, Enzymology method and boric acid are affine detects, and preferably employs Ai Kelai company of the Japan full-automatic glycolated hemoglobin analysis of HA-8180 in the present embodiment.
When, in glycosylation process, when the concentration of stable glycolated hemoglobin reaches required Quality Control substrate concentration, it is possible to use the red cell suspension after glycosylation is washed by cell washing solution, removing the mannose in this red cell suspension.When deglycosylation processes, it is necessary to diluent by erythrocyte suspendible.The present invention utilizes the cell washing solution of Washed Red Blood Cells, in fact can also utilize other known diluent, as long as not containing mannose in this diluent.Dilution ratio is minimum for 1:3, the i.e. ratio of red cell suspension and cell washing solution, for instance 1:5.And in deglycosylation process, erythrocyte is in the diluent suspendible regular hour, so that L-A1c is reduced to normal scope.Temperature is too low, and the deglycosylation time is oversize.If temperature is too high, it is possible to erythrocyte can be damaged.Therefore, deglycosylated temperature is preferably 18-23 DEG C.The deglycosylated time then determines according to the concentration of unstable glycolated hemoglobin L-A1c.In order to accurately determine the deglycosylation time, this deglycosylation process can also measure the concentration of L-A1c glycolated hemoglobin.Any analytical technology can be used to monitor the concentration of L-Alc glycolated hemoglobin, preferred high pressure liquid chromatographic analysis technology in the embodiment of the present invention.
When, in deglycosylation processing procedure, L-Alc glycolated hemoglobin reaches desirable concentration, erythrocyte and reactant liquor being easily separated, the embodiment of the present invention preferably employs centrifugal separation method.
S5, adds stabilizer, freeze drying protectant in deglycosylated red cell suspension, makes the glycolated hemoglobin Quality Control thing of liquid or lyophilizing.
Aforementioned stable agent may utilize the compositions listing composition in above-mentioned table 3, and during as stabilizer, adopts glucose to replace mannose.
The erythrocyte of embodiment of the present invention employing normal person carries out external glycosylation and carries out the preparation of glycolated hemoglobin Quality Control thing, expands the source of raw material.It addition, the embodiment of the present invention utilizes non-glucose hexahydroxylic alcohols hexose as external glycosylated saccharide, shorten the external glycosylated time.And, the embodiment of the present invention also utilizes cell washing solution to carry out deglycosylation process so that the concentration of glycolated hemoglobin unstable in the glycolated hemoglobin Quality Control thing after preparation reduces, thus ensure that the stability of glycolated hemoglobin Quality Control thing.
External glycosylation can use substantial amounts of normal healthy human blood's sample to produce the glycolated hemoglobin Quality Control thing of high glycolated hemoglobin concentration.Because these normal blood samples are from healthy individuality, they are typically free of hemoglobin variant variant.In addition external glycosylation process can also produce the Quality Control thing of the glycolated hemoglobin of variable concentrations as required to meet growing demand.
Further, as it is shown on figure 3, also include before above-mentioned steps S1:
S0, adds antioxidant in blood sample.
Utilizing antioxidant to contact with the hemoglobin in the erythrocyte of blood sample, the hemoglobin in erythrocyte can be prevented from ferrous iron Valence change to ferric iron valence state, Fe2+ is positioned at the center of hemoglobin, combines and carry oxygen in blood flow process.When Fe in hemoglobin is divalent ion state, erythrocyte erythrocyte retains redness.Generate ferric iron during bivalence Fe oxidation, make erythrocyte become sepia.Any such can stop the reagent of this oxidation to use.In the embodiment of the present invention, it is preferred to use carbon monoxide (CO) gas is as antioxidant.Making antioxidant contact with erythrocyte under certain condition, in erythrocyte, the ferrous iron of hemoglobin and antioxidant combine and make erythrocyte keep redness.The about 50%-80%Fe2+ of molar percentage combines with antioxidant or is connected.It is understood that before above-mentioned steps S0 can also be positioned at step S2, or antioxidant is added in cell washing solution, for erythrocyte is washed, form red cell suspension.Can join in cell detergent when antioxidant is in liquid or solid.
The preparation process of glycolated hemoglobin Quality Control thing will be illustrated below:
Thering is provided in a blood sample, in this blood sample, the concentration of hemoglobin is 120g/L, and erythrocytic concentration is 4*1012/L, and the concentration of stable glycolated hemoglobin is S-Alc:5-6%.First it is centrifuged this blood sample separating, generates erythrocyte, and in this blood sample, pass into the mode of CO bubble.Then utilize cell washing solution that this blood sample is washed, form cell suspension.Add reactant liquor, containing mannose 6% in this reactant liquor, 0.3%BSA phosphate buffer etc..Being placed at a set temperature by the cell suspension adding reactant liquor, the reaction setting time carries out glycosylation, and different time sections measures the concentration of glycolated hemoglobin simultaneously.When the concentration measuring glycolated hemoglobin reaches desired concn, mixture utilizes diluent (such as cell washing solution) carry out washing deglycosylation and processes.Erythrocyte mixture after glycosylation is washed 3 times, and diluent, containing 3% buffer, does not contain mannose.Typical washing process includes using diluent dilute sample, is thoroughly mixed, and is then centrifuged 15 minutes at 1500rpm, removes Aspirate supernatant after precipitating.Dilution ratio is 1:5.The buffer haemolysis of surfactant also can be added after deglycosylation, centrifugal segregation impurity, after adding freeze drying protectant, stabilizer, lyophilizing or liquid stored frozen.It addition, also can to volume be adjusted so that hemoglobin concentration remains 110-120g/L.
The present invention is by controlling the concentration of reactant liquor, reaction temperature and response time, it is possible to prepare the glycolated hemoglobin Quality Control thing of two or more concentration.Two groups or more glycolated hemoglobin can be assembled into external member.The concentration of usual one group of S-Alc glycolated hemoglobin is (non-diabetic range, about 4%-6%) within normal range, and the concentration of another group S-Alc glycolated hemoglobin is about 10-12%.
These are only the preferred embodiments of the present invention; not thereby the scope of the claims of the present invention is limited; every equivalent structure utilizing description of the present invention and accompanying drawing content to make or equivalence flow process conversion; or directly or indirectly it is used in other relevant technical fields, all in like manner include in the scope of patent protection of the present invention.
Claims (10)
1. the preparation method of a glycolated hemoglobin Quality Control thing, it is characterised in that comprise the following steps:
S1, is centrifuged blood sample processing, it is thus achieved that erythrocyte；
S2, utilizes cell washing solution that described erythrocyte is washed, and forms red cell suspension；
S3, carries out glycosylation in erythrocyte addition reactant liquor after washing；
S4, when the concentration of glycolated hemoglobin reaches required Quality Control substrate concentration in glycosylation process, utilizes cell washing solution that the red cell suspension of described step S3 is washed, carries out deglycosylation；
S5, adds stabilizer, freeze drying protectant in deglycosylated red cell suspension, makes the glycolated hemoglobin Quality Control thing of liquid or lyophilizing.
2. the preparation method of glycolated hemoglobin Quality Control thing as claimed in claim 1, it is characterised in that also include before described step S1:
S0, adds antioxidant in blood sample.
3. the preparation method of glycolated hemoglobin Quality Control thing as claimed in claim 1, it is characterised in that the composition of described cell detergent is:
Polyethylene glycol: 0～3%；
Disodium: 0～3%；
Magnesium gluconate: 0-1%；
Na2HPO4:0-2%；
Non-glucose hexahydroxylic alcohols hexose: 0-8%；
Methyl parahydroxybenzoate: 0-0.2%；
Inosine: 0-0.5%；
Neomycin: 0-0.2%；
Chloromycetin: 0-0.2%；
(NaOH): 0-0.5%；
(KCl): 0-1.5%；
Osmolarity: 250-300mOsm/L；
Above-mentioned solvent is deionized water.
4. the preparation method of glycolated hemoglobin Quality Control thing as stated in claim 3, it is characterised in that described non-glucose hexahydroxylic alcohols hexose is mannose.
5. the preparation method of glycolated hemoglobin Quality Control thing as claimed in claim 1, it is characterised in that the described composition containing mannose or the reactant liquor of glucose is:
Polyethylene glycol (200-50000): 0-3%；
Disodium: 0-3%；
Magnesium gluconate: 0-1%；
Na2HPO4:0-2%；
Mannose or glucose: 0-8%；
Methyl parahydroxybenzoate: 0-0.2%；
Inosine: 0-0.6%；
Neomycin: 0-0.2%；
Chloromycetin: 0-0.2%；
(NaOH): 0-0.05%；
(KCl): 0-1.5%；
NaF:0～0.5%；
Ph:6.0～8.0；
Osmolarity: 250-300mOsm/L；
Above-mentioned solvent is deionized water.
6. the preparation method of glycolated hemoglobin Quality Control thing as claimed in claim 4, it is characterised in that be additionally added bovine serum albumin in described reactant liquor, and the mass concentration that described bovine serum albumin is in reactant liquor is 1～6%.
7. the preparation method of glycolated hemoglobin Quality Control thing as claimed in claim 1, it is characterised in that reaction temperature when carrying out glycosylation in described step S3 is 18～23 DEG C, and the response time is 4～8 days.
8. the preparation method of glycolated hemoglobin Quality Control thing as claimed in claim 1, it is characterised in that in described blood sample, erythrocytic concentration is: 3.5*1012/L-5.0*1012/L, and the concentration of hemoglobin is: 9g/L-12g/L.
9. the preparation method of glycolated hemoglobin Quality Control thing as claimed in claim 1, it is characterised in that in described step S4, adds the red cell suspension after glycosylation cell washing solution, and carries out being centrifuged off precipitation, leave and take supernatant.
10. the preparation method of glycolated hemoglobin Quality Control thing as claimed in claim 9, it is characterised in that described red cell suspension is minimum for 1:3 with the ratio of cell washing solution.
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| CN109239065A (en) * | 2018-09-06 | 2019-01-18 | 迪瑞医疗科技股份有限公司 | A kind of stable urine total protein quality-control product and its preparation method and application |
| CN109269858A (en) * | 2018-11-02 | 2019-01-25 | 郑州标源生物科技有限公司 | A kind of preparation method of liquid saccharified hemoglobin quality-control product |
| CN109596846A (en) * | 2018-11-22 | 2019-04-09 | 东软威特曼生物科技(南京)有限公司 | It is a kind of for external diagnosis reagent calibration object, the compound stabilizer of quality-control product |
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| CN106771261A (en) * | 2017-01-20 | 2017-05-31 | 深圳市雷诺华科技实业有限公司 | The preparation method and its quality-control product of glycosylated hemoglobin quality-control product |
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| CN109239065A (en) * | 2018-09-06 | 2019-01-18 | 迪瑞医疗科技股份有限公司 | A kind of stable urine total protein quality-control product and its preparation method and application |
| CN109239065B (en) * | 2018-09-06 | 2021-09-28 | 迪瑞医疗科技股份有限公司 | Stable urine total protein quality control product and preparation method and application thereof |
| CN109269858A (en) * | 2018-11-02 | 2019-01-25 | 郑州标源生物科技有限公司 | A kind of preparation method of liquid saccharified hemoglobin quality-control product |
| CN109596846A (en) * | 2018-11-22 | 2019-04-09 | 东软威特曼生物科技(南京)有限公司 | It is a kind of for external diagnosis reagent calibration object, the compound stabilizer of quality-control product |
| CN109856409A (en) * | 2019-01-03 | 2019-06-07 | 桂林优利特医疗电子有限公司 | The application of dry type examples of hemoglobin detection system hemoglobin control liquid |
| CN109856409B (en) * | 2019-01-03 | 2023-03-31 | 桂林优利特医疗电子有限公司 | Application of hemoglobin control liquid for dry hemoglobin detection system |
| CN111024959A (en) * | 2019-12-20 | 2020-04-17 | 深圳市蔚景生物科技有限公司 | Stable protein solution, preparation method thereof and detection kit |
| CN114112587A (en) * | 2021-11-25 | 2022-03-01 | 青岛汉唐生物科技有限公司 | Preparation method of glycated albumin calibrator or quality control product |
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