Hemoglobin can also bind glucose to one or both of the NH2-terminal valine residues of the β- globin chain. Glucose first becomes attached via a non-enzymatic process, in a reversible binding that leads to the formation of unstable aldimine, followed by a rearrangement to form an irreversible ketoamine. These adducts are named glycohemoglobins, glycated hemoglobin, glycosylated hemoglobin, or - most often - HbA1c. Hemoglobin that is formed in the bone marrow enters the circulation in the erythrocyte without any attached glucose, but the red blood cell is permeable for glucose once it reaches the circulation. Glucose therefore becomes attached to hemoglobin during the entire life span of the blood cell, or about 120 days. The degree of glucose binding depends on the prevailing blood glucose. As the fraction of HbA1c of total hemoglobin, expresses the mean value from all red blood cells, it changes in a dynamic way and reflects the Mean Blood Glucose over the previous 6-8 weeks. It can be said to be a “weighted” average of blood glucose.


What is HbA1c?
HbA1c designates a stable minor glycated sub fraction of hemoglobin. It is a reflection of the mean blood glucose levels during the last 6-8 weeks, and is expressed in % of total hemoglobin. HbA1c does correlate well with average blood glucose, but does not say anything about the blood glucose fluctuations. An HbA1c value of 7.5% will, however, correspond to fasting blood glucose values of 5-8 mmol/L or a mean blood glucose value of somewhat lower than 10 mmol/L. The first clinically useful method for determining of HbA1c was described in 1978. Since then about 20 methods of determination of HbA1c, based on HPLC, electrophoresis, immunology and affinity methods have been developed. Different methods, however, do not measure identical adducts. Some methods measure all adducts, others not. Therefore values have to be standardized, and international standardisation work is well underway. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) have presented an approved reference method for the measurement of HbA1c. It is the basis for the uniform standardization of HbA1c routine assays worldwide.

Another problem associated with HbA1c determination and interpretation, is associated with racial/ethnic genetic variations affecting the binding of glucose to hemoglobin. These genetic variations affect different sites at the hemoglobin molecule, and can therefore influence values obtained by the various methods to different degree, as the different methods do not measure the same adducts. As a result some methods can give false high or false low HbA1c in subjects carrying such mutations, and therefore be misleading. As the prevalence of such mutations varies greatly with ethnicity and thus geographically, the choice of method, and interpretation of results should be based on knowledge and identification of locally common hemoglobinopathies, and how they influence different assays of HbA1c. This problem was recently highlighted by data from the National Health and Nutrition Examination Survey, in which HbA1c levels were significantly higher in blacks and Hispanics than in whites. Screening and intervention therefore needs to consider racial and ethnic disparities in order to optimise diagnosis and treatment of diabetes.


Hemoglobin variants
When organisms reproduce, they copy their DNA. Occasionally mistakes occur that may be random errors during copying, or the result of toxic products or radiation, causing mutations in the DNA of the following generations. More than 700 mutant variations of hemoglobin have been identified in humans, evidence of an ongoing evolution of hemoglobin genes. Most variations exist only in a small fraction of populations, but some are quite frequent in certain populations. A few of these mutant forms cause disease, especially in homozygous state. Sickle-cell hemoglobin is the most infamous. Other variants do not give any symptoms, and a very few may even have advantages. Even if these mutations are otherwise harmless, they may, however, interfere with assays for HbA1c.

Normally, the hemoglobin consists of about 97% HbA, 2.5% HbA2, and 0.5% HbF. Structural variants of hemoglobin contain genetically determined changes in the primary structures of the α-, β-, γ- and δ-chains, from which these forms are assembled; HbA (α2 β2), HbF (α2 γ2) and HbA2 (α2 δ2). Thalassemias are conditions in which there is a reduction in the rate of synthesis of one type of globin chain. α-Thalassemia is the most common genetic disorder. About 30% of black Americans have a gene for α-thalassemia. It is about as frequent in Southeast Asia. β -Thalassemia and hereditary persistent HbF (HPHF) are caused by impaired production of β-chains. β –Thalassemia is frequent in Mediterranean and Asian populations. Blood from these patients will contain increased levels of HbF. In HPHF of African type HbF usually constitutes 15-30% of total hemoglobin. In patients with the rare sickling disorder (homoxygous) the blood will contain only 10-25% HbA, the rest being HbS.

The variants hemoglobin HbS, HbC and HbE are containing point mutations in the β-chain; HbS (β6 glu→val), HbC (β6 glu→lys) and HbE (β26 glu→lys).
These mutations may interfere with HbA1c determinations more or less, depending on the technique used. HPLC methods, for instance, do not recognise the glycated fractions of certain variants and may therefore give false low HbA1c. Moreover different variant peaks may overlap or coincide with the HbA1c peak and give false high results. Electrophoresis is also unsuitable for patients with homozygous variants, as this technique does not separate HbA0 from HbE0 or HbA1c from HbE1c. Therefore, the value will increase in patients with HbAE. HbF will also cause falsely high value. For immunological methods that recognise the different fractions there will be no interference in contrast to methods that use antibodies specific for HbA1c. Boronate affinity methods, on which AfinionTM HbA1c and NycoCardâ HbA1c are based, measure all the glycated components, and have been shown to indicate values in an acceptable range also for patients with a range of different mutations.