1. Cation exchange chromatography
Principle: Glycation results in the loss of cations from the surface of the hemoglobin molecule. In a weak cation exchanger, such as Biorex70, accompanied by an increase in ion concentration and/or a decrease in pH, glycated hemoglobin elutes before non-glycated hemoglobin. This phenomenon gave rise to the original term "fast hemoglobin" for glycated hemoglobin. Cation exchange chromatography can be used in small, micro or large column chromatography methods or in partially or fully automated PHLC/FPLC methods. Because other post-translational modifications of hemoglobin, such as aldimine-type, formylation, acetylation, acetaldehyde adducts, degradation products, aging artifacts, and abnormal hemoglobin charge exchanges also differ from normal HbA0, a number of interferences with cation-exchange chromatography methods have been listed. Methods using conventional HPLC. Separation of glycosylated hemoglobin subfractions is capable of achieving the clinical precision needed to meet the demand. However, it is known that the peak of HbA1c is not homogeneous but contains a significant non-glycosylated hemoglobin fraction. A small amount of glycated hemoglobin is also integrated into the main HbA0 peak. The separation can be improved by using a special column material (poly-CATA) and a separation time of 30-40 min. These methods can be used as reference steps but are not suitable for routine use. All cation exchange chromatography methods are sensitive to changes in pH and temperature, so control pH and temperature.
Note: Initial HbA1c values are presumed to be destroyed approximately 1/120 (≈0.83%) per day based on erythrocyte metabolic kinetics. This theoretical value cannot be reached in vitro because glycation is produced even in healthy individuals under appropriate treatment. In diabetic patients with suboptimal control who achieve a normal glycemic load by intensified therapy, the maximum rate of decrease in HbA1c values can be found at a rate of approximately 1% of normal blood glucose (absolute) every 10 d. The rate of decrease in HbA1c values is also higher in diabetic patients with suboptimal control than in healthy patients. Because of the precision of the method of determining glycosylated hemoglobin, a difference of approximately 1% between two measurements of HbA1c can be considered clinically relevant. For these reasons, a minimum of 2 weeks should be allowed between HbA1c measurements, with a recommended interval of 4 to 6 weeks.
Because elevated glycosylated hemoglobin values are a fairly reliable indicator in diabetic patients with prolonged hyperglycemia and are therefore potentially diagnostic of diabetes. In untreated individuals, a normal glycosylated hemoglobin value clinically rules out overt diabetes. However, because it does not detect impaired glucose tolerance, the use of glycosylated hemoglobin as the only parameter for diagnostic and/or screening purposes is problematic.
2. Electrophoresis
Principle: Compared with non-glycosylated hemoglobin, the change in total charge due to glycation and the change in the isoelectric point of glycosylated hemoglobin are the basis for isoelectrofocused electrophoretic separation on agarose gels or gels with pH gradients of 5.0 to 6.5. Agarose gel electrophoresis has a small resolution of hemoglobin subfractions, and isoelectric focusing allows better separation of subfractions. The importance has declined, probably due to the lack of automation of the test.
3. Affinity chromatography
Principle: Boric acid binds cis-hydroxyl. Commercial m-aminophenylboronic acid agarose *** valence-bound affinity columns have been available for microcolumn analytical detection. Upon addition of hemoglobin from a blood sample to the column, all glycosylated hemoglobin (HbA1 and side-chain glycosylated hemoglobin; total glycosylated hemoglobin) is bound to the boric acid while the non-glycosylated hemoglobin passes through the column and can be measured. Upon addition of a high concentration of a polyhydroxyl complex that also contains cis-hydroxyl groups, such as sorbitol, the binding of glycated hemoglobin to boric acid is replaced and elutes from the column. Affinity chromatography is relatively insensitive to the effects of hemoglobins modified by post-translational modifications and pathological hemoglobins. Using affinity chromatography, only total glycosylated hemoglobin can be determined. The widely used affinity chromatography method allows an empirical algorithm to calculate a "standard HbA1c" from the total glycated hemoglobin value.
4. Immunoassay
Hemoglobin glycosylated at the valine β-N-terminus provides an antigenic epitope that is readily recognized by antibodies. It can be determined by radioimmunoassay and immunoenzymatic analysis using monoclonal or polyclonal antibodies, which specifically recognize the antigenic epitope consisting of the last 4 to 8 amino acids of the β-chain N-terminally glycosylated hemoglobin. Abnormal hemoglobins or post-translationally modified hemoglobins did not interfere.
Current immunochemical tests detect not only HbA1c, but usually also HbA2c at the same time, because the epitopes of the glycosylated δ-chain of hemoglobin A2 are identical. Immunochemical tests in which the antibody is directed against the glycosylated epitopes of the last four amino acids of the β-chain can also be used to detect, for example, HbS1c. In most cases HbA2c is not very significant, and although the degree of valine β-N-amino-terminal glycosylation can be measured accurately in sickle cell disease, it still does not represent 100% of the HbA1c.
5、Ion chromatography
This is a highly precise and accurate test. Ion chromatography is a highly precise, reproducible and simple method that is widely used in clinical practice. The principle of the assay is due to the different charges of hemoglobin β-chain N-terminal valine glycation, total glycated hemoglobin (GH b) and H bA in acidic solution have cationic properties, so when passing through the cation exchange chromatography column, it can be adsorbed by the resin balanced by acidic buffer, but the adsorption rate of the two is different, the positive charge of GH b is lower, the lower adsorption rate, and the higher rate of adsorption of the more positive charge of H bA. GH b and H bA can be eluted with different pH phosphate buffers, and KCN can be used to convert H b to ferricyanide hemoglobin, which can be measured spectrophotometrically. Alternatively, the corresponding Hb chromatographic spectrum can be obtained, with the horizontal coordinate being the time and the vertical coordinate being the percentage.HbA1c values are expressed as a percentage. HbA1c values are expressed as a percentage. Nowadays, most of them are measured with a fully automated meter.
6, isoelectric point aggregation method
is the determination of GH b of the new technology, it is in the polypropylene phthalide gel with a carrier amphoteric medium on the formation of a gradient from the anode to the cathode of the pH gradient, soluble blood of the components will be moved to their respective isoelectric point of the pH position, so that better than the general electrophoresis method of the delineation of the effect of the ribbon and the more concentrated, through the high resolution micro-light densitometer, the chromatographic spectrum. This results in a better delineation and more concentrated color bands than normal electrophoresis, which can be accurately determined by scanning with a high-resolution micro-optical densitometer. Since it can distinguish HbA, HbAc, HbF, HbS and HbC with different primary structures, the interference of various substances can be completely avoided.
7, chemiluminescence method
Ion capture immunoassay, the application of the principle of antigen-antibody reaction, coupled with fluorescent markers, through the connection of negatively charged multi-anion complex, adsorption to the surface of the positively charged fibers, after a series of steps such as a thorough cleaning, the rate of change of the fluorescence intensity is measured, and the concentration is calculated. With the special reagent kit and immunoluminescence analyzer, the detection system is easy to be standardized and reproducible, which can reduce the operation technical error, high sensitivity and specificity of the test, small coefficient of variation between batches and within a batch, high recoveries, high accuracy, small cross-contamination rate, and fewer influencing factors.
8, enzyme method
Principle is to use special protease to decompose Hb, 3~5 min fructosyl amino acid from H b separation, fructosyl amino acid oxidase ( FAOD ) from the fructosyl amino acid to produce H2O2, H2O2 by POD and DA-64 reaction, choose 751 nm absorbance change to find the GHb concentration.