The clinical value and detection method of glycosylated
2019-05-22
Diabetes mellitus is a group of endocrine and metabolic diseases whose etiology and pathogenesis have not been completely understood. At present, the incidence of diabetes is only second to cardiovascular and cerebrovascular diseases and tumors. The incidence of diabetes mellitus in China is 2% ~ 3%, and it is increasing at the rate of 1% per year.
At present, clinical blood glucose testing has been widely carried out in patients.
However, the blood glucose measurement only represents the immediate blood glucose level, indicating the patient's physical condition at that time, and cannot be used as an indicator to evaluate the degree of disease control.
Therefore, it has long been a problem to evaluate the long-term control level of diabetes, especially for patients with fluctuating conditions and insulin injection.
Glycosylated hemoglobin (HbA1c) and red blood cells of glucose in the blood of the combination of the hemoglobin, blood glucose and hemoglobin generated glycosylated hemoglobin is irreversible reaction, and is proportional to the concentration of blood glucose, and keep 120 days or so, so of HbA1c concentrations in the blood can reflect the recent 2-3 months in average blood glucose levels.
It is the gold standard to evaluate the level of blood glucose control.
The characteristics of glycosylated hemoglobin determine its great significance in diabetes monitoring :(1) it is parallel to the blood glucose value.
The higher the blood sugar, the higher the hemoglobin a1c, so it reflects the level of blood sugar control.
(2) slow generation.
Since the blood glucose is constantly fluctuating, each blood drawing can only reflect the blood glucose level at that time, while the glycosylated hemoglobin is gradually generated, and the transient increase of blood glucose will not cause the increase of glycosylated hemoglobin.
In turn, a brief drop in blood sugar does not cause a drop in hemoglobin a1c.
Since eating does not affect the measurement, it can be measured after the meal.
(3) once generated, it is not easy to decompose.
Hba1c is fairly stable and not easily decomposed, so although it cannot reflect short-term blood glucose fluctuations, it can well reflect the degree of blood glucose control over a long period of time. Hba1c can reflect the average blood glucose level within 2 months before blood collection.
(4) less affected by hemoglobin levels.
The measurement of hba1c is expressed as a percentage, which refers to the proportion of hemoglobin bound to glucose in total hemoglobin.
Control criteria for hba1c
Hemoglobin a1c can reflect the average level of blood glucose control in the past 2-3 months, and it is not affected by the occasional rise or decrease of blood glucose. Therefore, the measurement of hemoglobin a1c can provide a more comprehensive understanding of the blood glucose control level in the past period.
World authority has clear control target for glycosylated hemoglobin, ADA (American diabetes association) suggest glycosylated hemoglobin control in less than 7%, the IDF (the international diabetes federation) suggest glycosylated hemoglobin control standard is less than 6.5%, at present, the control of the diabetes glycated hemoglobin standard was below 6.5%.
The level of hba1c in non-diabetic patients was 4-6%.
Many studies have found that people with diabetes, such as check can glycosylated hemoglobin levels below 8%, the complications of diabetes will be greatly reduced, if a1c > 9%, patients with permanent high blood sugar, diabetic kidney disease happens, hardening of the arteries, and complications such as cataract, and likely acute complications such as ketoacidosis.
It is of great significance for the prevention of macrosomia, malformation, stillbirth, and the occurrence and development of acute and chronic complications in pregnant women with diabetes.
For patients with undetermined etiology who are in coma or who are undergoing glucose infusion (of course the blood glucose is elevated), the urgent examination of hba1c has the value of differential diagnosis.
Diabetic patients with especially high glycosylated hemoglobin should be alert to the occurrence of acute complications such as ketoacidosis.
Therefore, relevant experts suggest that if the blood glucose control of diabetic patients has reached the standard, and the blood glucose control state is relatively stable, they should receive at least two times of hba1c testing every year.
Hba1c should be measured every three months in patients who need to change their data protocol, or whose blood glucose control is unstable, or who are on insulin treatment.
There are many methods to detect hba1c, including ion exchange high performance liquid chromatography (HPLC) and electrophoresis (electrophoresis), and affinity chromatography (affinity chromatography) and immunoassay (immunoassay) according to the charge difference between GHb and non-ghb.
Ion exchange high performance liquid chromatography
Based on the different charge of valine glycation at the n-terminal of Hbb chain.
Under neutral pH conditions,HbA1c has a relatively small positive charge, so it can be distinguished from other components (HbA1c,HbA1b, HbA1a,HbF,HbA0) by HPLC, and thus be separated.
This method has been used in the United States DCCT research, is the current gold standard for HbA1c detection.
electrophoresis
Colloidal particles can have charge under certain conditions. Colloidal particles with charge can swim in the electric field by electrostatic attraction. Those with positive charge swim to the negative pole and those with negative charge swim to the positive pole.
All kinds of proteins in serum have their unique isoelectric point, and all kinds of proteins are neutral at their isoelectric point. The positive and negative electric charges of their molecules are equal.
But the weaknesses of this methodology are:
1. Samples need to be analyzed in batches, which is slow and cannot be tested in real time
2. Low degree of automation, insufficient emergency insertion, automatic maintenance and other functions.
Affinity chromatography
The affinity adsorption system was prepared by using the principle of reversible binding of the biomolecule with the corresponding specific ligand molecule, which was firmly bonded to the solid phase carrier by covalent bond.
Under certain conditions, the macromolecular separation target with impurities can be combined with the already solid ligand by some secondary bonds, while the impurities are not adsorbed. After removing the impurities, the conditions can be changed, and the macromolecular materials to be separated can be dissociated again and purified.
All sugars in the blood can bind to Hb except glucose, so the test results for this method are total HbA1c, not HbA1c.
immunization
Monoclonal or polyclonal antibodies are used for the direct determination of 4-8 amino acids at the n-terminus glycosylated on the hemoglobin -chain and are therefore highly specific to the glycosylated HbA1C.
Mainly used in large biochemical analyzer, the advantage is that do not need to buy another instrument, simple operation, fast speed, high degree of automation.
But many homologous hba1c variants also have the same n-terminal structure, which can interfere with the results.
In the calibration, it is necessary to make relative correction with HPLC.
The HbA1C determination method commonly used in clinical laboratories is integrated. Due to different principles, the accuracy and repeatability of the determination results are different.
It is believed that with the emphasis and improvement on the standardization of HbA1c experimental methods, the detection of HbA1c project will be increasingly applied in clinical practice.
At present, clinical blood glucose testing has been widely carried out in patients.
However, the blood glucose measurement only represents the immediate blood glucose level, indicating the patient's physical condition at that time, and cannot be used as an indicator to evaluate the degree of disease control.
Therefore, it has long been a problem to evaluate the long-term control level of diabetes, especially for patients with fluctuating conditions and insulin injection.
Glycosylated hemoglobin (HbA1c) and red blood cells of glucose in the blood of the combination of the hemoglobin, blood glucose and hemoglobin generated glycosylated hemoglobin is irreversible reaction, and is proportional to the concentration of blood glucose, and keep 120 days or so, so of HbA1c concentrations in the blood can reflect the recent 2-3 months in average blood glucose levels.
It is the gold standard to evaluate the level of blood glucose control.
The characteristics of glycosylated hemoglobin determine its great significance in diabetes monitoring :(1) it is parallel to the blood glucose value.
The higher the blood sugar, the higher the hemoglobin a1c, so it reflects the level of blood sugar control.
(2) slow generation.
Since the blood glucose is constantly fluctuating, each blood drawing can only reflect the blood glucose level at that time, while the glycosylated hemoglobin is gradually generated, and the transient increase of blood glucose will not cause the increase of glycosylated hemoglobin.
In turn, a brief drop in blood sugar does not cause a drop in hemoglobin a1c.
Since eating does not affect the measurement, it can be measured after the meal.
(3) once generated, it is not easy to decompose.
Hba1c is fairly stable and not easily decomposed, so although it cannot reflect short-term blood glucose fluctuations, it can well reflect the degree of blood glucose control over a long period of time. Hba1c can reflect the average blood glucose level within 2 months before blood collection.
(4) less affected by hemoglobin levels.
The measurement of hba1c is expressed as a percentage, which refers to the proportion of hemoglobin bound to glucose in total hemoglobin.
Control criteria for hba1c
Hemoglobin a1c can reflect the average level of blood glucose control in the past 2-3 months, and it is not affected by the occasional rise or decrease of blood glucose. Therefore, the measurement of hemoglobin a1c can provide a more comprehensive understanding of the blood glucose control level in the past period.
World authority has clear control target for glycosylated hemoglobin, ADA (American diabetes association) suggest glycosylated hemoglobin control in less than 7%, the IDF (the international diabetes federation) suggest glycosylated hemoglobin control standard is less than 6.5%, at present, the control of the diabetes glycated hemoglobin standard was below 6.5%.
The level of hba1c in non-diabetic patients was 4-6%.
Many studies have found that people with diabetes, such as check can glycosylated hemoglobin levels below 8%, the complications of diabetes will be greatly reduced, if a1c > 9%, patients with permanent high blood sugar, diabetic kidney disease happens, hardening of the arteries, and complications such as cataract, and likely acute complications such as ketoacidosis.
It is of great significance for the prevention of macrosomia, malformation, stillbirth, and the occurrence and development of acute and chronic complications in pregnant women with diabetes.
For patients with undetermined etiology who are in coma or who are undergoing glucose infusion (of course the blood glucose is elevated), the urgent examination of hba1c has the value of differential diagnosis.
Diabetic patients with especially high glycosylated hemoglobin should be alert to the occurrence of acute complications such as ketoacidosis.
Therefore, relevant experts suggest that if the blood glucose control of diabetic patients has reached the standard, and the blood glucose control state is relatively stable, they should receive at least two times of hba1c testing every year.
Hba1c should be measured every three months in patients who need to change their data protocol, or whose blood glucose control is unstable, or who are on insulin treatment.
There are many methods to detect hba1c, including ion exchange high performance liquid chromatography (HPLC) and electrophoresis (electrophoresis), and affinity chromatography (affinity chromatography) and immunoassay (immunoassay) according to the charge difference between GHb and non-ghb.
Ion exchange high performance liquid chromatography
Based on the different charge of valine glycation at the n-terminal of Hbb chain.
Under neutral pH conditions,HbA1c has a relatively small positive charge, so it can be distinguished from other components (HbA1c,HbA1b, HbA1a,HbF,HbA0) by HPLC, and thus be separated.
This method has been used in the United States DCCT research, is the current gold standard for HbA1c detection.
electrophoresis
Colloidal particles can have charge under certain conditions. Colloidal particles with charge can swim in the electric field by electrostatic attraction. Those with positive charge swim to the negative pole and those with negative charge swim to the positive pole.
All kinds of proteins in serum have their unique isoelectric point, and all kinds of proteins are neutral at their isoelectric point. The positive and negative electric charges of their molecules are equal.
But the weaknesses of this methodology are:
1. Samples need to be analyzed in batches, which is slow and cannot be tested in real time
2. Low degree of automation, insufficient emergency insertion, automatic maintenance and other functions.
Affinity chromatography
The affinity adsorption system was prepared by using the principle of reversible binding of the biomolecule with the corresponding specific ligand molecule, which was firmly bonded to the solid phase carrier by covalent bond.
Under certain conditions, the macromolecular separation target with impurities can be combined with the already solid ligand by some secondary bonds, while the impurities are not adsorbed. After removing the impurities, the conditions can be changed, and the macromolecular materials to be separated can be dissociated again and purified.
All sugars in the blood can bind to Hb except glucose, so the test results for this method are total HbA1c, not HbA1c.
immunization
Monoclonal or polyclonal antibodies are used for the direct determination of 4-8 amino acids at the n-terminus glycosylated on the hemoglobin -chain and are therefore highly specific to the glycosylated HbA1C.
Mainly used in large biochemical analyzer, the advantage is that do not need to buy another instrument, simple operation, fast speed, high degree of automation.
But many homologous hba1c variants also have the same n-terminal structure, which can interfere with the results.
In the calibration, it is necessary to make relative correction with HPLC.
The HbA1C determination method commonly used in clinical laboratories is integrated. Due to different principles, the accuracy and repeatability of the determination results are different.
It is believed that with the emphasis and improvement on the standardization of HbA1c experimental methods, the detection of HbA1c project will be increasingly applied in clinical practice.