Introduction

Glycated hemoglobin (HbA1c) analysis is a crucial component in the management of diabetes mellitus. It provides a long-term assessment of an individual’s average blood glucose levels over the preceding 2-3 months, making it an essential tool for both diagnosis and monitoring of diabetes. To perform accurate HbA1c analysis, specialized instruments known as glycated hemoglobin analyzers are employed. This article elucidates the principles and methods of HbA1c analysis using such an analyzer.

Principles of HbA1c Analysis

No matter which measurement principle is used, sample pretreatment is required, including the removal of other interfering substances, such as HbF, HbS, etc., to ensure the accuracy of the results.

The glycated hemoglobin automatic analyzer uses the ion exchange HPLC method to measure glycated hemoglobin (HbA1c). Ion exchange HPLC is a method that uses materials that can exchange ions as stationary phases to separate ionic compounds. The instrument uses a cation exchange column to measure the percentage of HbA1c; when a certain amount of whole blood sample is sucked into the sampling device by the sampling needle, it is hemolyzed in the diluted part to release the hemoglobin Hb in the red blood cells, and is diluted with the diluent. The hemolyzed sample is injected into the ion exchange column by a high-pressure pump. After the sample is added from the top of the exchange column through the filter, it is eluted by three different concentrations of salt elution buffer (mobile phase), causing the sample to move downward. At this time, various components of hemoglobin Hb contained in the solution are exchanged with movable ions on the stationary phase. The various components of hemoglobin in the sample continuously undergo reversible exchange adsorption and desorption on the stationary phase, and the three types Salt solutions with different ion concentrations form a linear gradient elution.

The fully automatic hemoglobin analyzer separates various components in Hb, among which HbA1c, HbF, and HbA1 are effectively and accurately separated; the Hb components flowing out from the exchange column reach the detector of the instrument, which is equipped with a luminescent device that emits monochromatic light. Diode, measure the three parameters of HbA1c, HbF and HbA1 through dual-wavelength visible light colorimetry. The detector of the instrument detects the absorbance values of the HbA1c, HbF, and HbA1 components after separation, compares them with the absorbance values of the HbA1c standard, and analyzes and calculates the results.

Methods of HbA1c Analysis

Glycated hemoglobin (HbA1c) is the product of the combination of hemoglobin in red blood cells and sugars (mainly glucose) in the serum through a non-enzymatic reaction. The non-enzymatic reaction that forms glycated hemoglobin is continuous, slow, and irreversible. It is generally believed that glycated hemoglobin concentration can effectively reflect the average blood sugar level in the past 8 to 12 weeks. Glycated hemoglobin is composed of HbA1a, HbA1b, and HbA1c, of which HbA1c accounts for about 70% and has a relatively stable structure. It is often used clinically as a monitoring indicator for diabetes control. Its concentration should be expressed as a percentage of adult hemoglobin.

Determination methods used in clinical and laboratory applications are mainly divided into two categories:

One is based on the different charges of glycated hemoglobin and non-glycated hemoglobin, such as ion exchange chromatography, electrophoresis and isoelectric aggregation methods.

The second is based on the structural characteristics of the glycated groups on hemoglobin, such as affinity chromatography and immunological experiments.

(1) Affinity chromatography: This method has strong specificity and is not interfered by abnormal hemoglobin. It is suitable for use in clinical departments, especially for pediatric patients. The test results also fully meet and exceed clinical requirements, with a CV value within 5%.

(2) High-pressure liquid chromatography method (HPLC) can fully automatically separate and measure glycosylated hemoglobin and blood protein variants and subtypes, but the operation and maintenance requirements of the instrument are relatively high, and the CV value is within 1%.

(3) Immunoagglutination method: The principle is that glycated hemoglobin binds to the corresponding monoclonal antibody and agglutination reaction occurs. The amount of agglutination is expressed by measuring the absorbance, which can be used for measurement on a fully automatic biochemical analyzer. A new kit should be used for each test, and reagents should be mixed well before operation. It is pointed out that the immunoagglutination method to measure glycated hemoglobin has poor precision, and the CV value is generally less than 5%.

(4) Ion capture method: The principle is that after glycated hemoglobin is combined with the corresponding antibody, it is coupled with a fluorescent marker, and the glass fiber in the IMX reaction well is pre-coated with a polymer tetraamine compound to make the surface of the fiber positively charged. Electricity causes the aforementioned reaction complex to be adsorbed on the surface of the fiber. After a series of cleanings, its fluorescence intensity is measured to obtain the concentration of glycated hemoglobin. This method is suitable for the detection of batches of glycated hemoglobin specimens.

(5) Electrophoresis method: Capillary electrophoresis can also separate and detect glycosylated hemoglobin and hemoglobin variants, but it has not yet been commercialized. The availability of instruments with the ability to pass batch samples has greatly limited the clinical application of this method.

Conclusion

Glycated hemoglobin analysis is a fundamental tool for the management of diabetes, enabling healthcare professionals to assess long-term glycemic control in patients. Glycated hemoglobin analyzers, using principles of high-performance liquid chromatography, provide accurate and reliable results. Understanding the principles and methods behind these analyzers is crucial for healthcare practitioners to make informed decisions regarding patient care and treatment adjustments in diabetes management.

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