Academy of Diagnostics & Laboratory Medicine - Scientific Short
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CDC statistics (2023) reported that ~8.9% of the USA population is affected by DM and about 38% of the adults are in the high-risk for developing diabetes. In the last 15 years the number of adults suffering from DM has almost tripled and the trend is on the rise. The groups with the highest incidence of DM are adults over the age of 45 and individuals belonging to demographic groups with high prevalence (e.g. Blacks, Hispanics, and Native Americans).
While glucose evaluations can provide only time-limited information about the glycemic level, evaluation of HbA1c provides further advantages (Table 1). (1, 2). As such, the ADA recommends HbA1c as an indicator of long-term glycemic control in patients with DM, as well as for the screening and diagnosis of DM, with a recommended cutoff of 6.5%.
Table 1: Comparison of glucose and HbA1c testing modality advantages and limitations.
| Glucose evaluation | HbA1c evaluation |
| Fasting | No fasting necessary |
| Multiple draws (e.g. OGTT) | One draw |
| Reflects immediate glycemic level | Reflects long-term glycemic level (2-3 months) |
| No hemoglobin variants interferences | Hb variants can interfere |
HbA1c is produced by nonenzymatic addition of a glucose molecule to the N-terminal of the β chain of Hb A. Glycation of the N-terminal residue changes its structure and decreases the positive charge of Hb A. (3)
Methods of HbA1c analysis can be divided into 2 categories: methods based on molecular charge and those based on structure. The former category includes CE-HPLC (Bio-Rad) and electrophoresis (Sebia Capillarys), and the latter includes immunoassays (various manufacturers), boronate affinity chromatography (Trinity Biotech), and mass spectrometry (3).
In CE-HPLC and electrophoresis assays, HbA1c can be separated from Hb A because glycation decreases the positive charge. Therefore, charge-based methods may be affected by posttranslational modifications (e.g., carbamylation and acetylation) (3) or by Hb mutations (variants) (3) that alter hemoglobin charge. Over 1000 hemoglobin variants (alpha-, beta-, gamma- or delta-chains) have been described, and, depending on their charge, they can interfere with accurate evaluation of HbA1c when using these charge-based methods. Despite of these, ADA considers CE-HPLC the reference method for evaluation of HbA1c.
Immunoassays use antibodies that target N-terminal glycated amino acids on the β chain to quantify HbA1c , and the HbA1c percentage is calculated from the HbA1c and the total Hb concentrations (3). Any factor that prevents glycation or any mutation in the epitope region can affect antibody recognition and produce erroneous results. Additionally, patients with increased Hb F (>10%, e.g. sickle cell patients treated with hydroxyurea or patients with HPHF) are prone to falsely low HbA1c value by immunoassay. This is due to the fact that, although total Hb concentration is included in the calculation of the %HbA1c, HbF γ chains share only 4 of the first 10 amino acids with the β chain of Hb A and has little to no immunoreactivity with most antibodies used in HbA1c assays (3).
In the boronate affinity chromatography (BAC), boronic acid reacts with the cis-diol groups created by glycation, HbA1c to be very specifically separated from Hb A (3). Due to the design of the assay, BAC is virtually free of interferences form hemoglobin variants. However, elevated HbF can still lead to spurious results due to reasons mentioned above.
Mass spectrometric assay, an IFCC reference method, specifically measures the glycated N-terminal valine of the Hb A β chain (3), but the prohibitive cost of a mass spectrometer and the complicated nature of its installation and operation are likely to preclude its use in most clinical laboratories in the near future (3).
When using any of the analytical methods available on the market, common hemoglobin variants traits do not generally interfere with the HbA1c evaluation. However, this is not valid in the case of certain hemoglobin variants, especially when charge-based and IA laboratory assessments are used. (3)
In patients with both -globin alleles affected by mutation, such as homozygous mutations (e.g. HbSS-sickle cell disease and HbCC-hemoglobin C disease), or compound heterozygous mutations (e.g. HbSC), although the process of hemoglobin glycation still occurs, leading to formation of glycated variant hemoglobins, these patients do not produce normal HbA and therefore neither HbA1c.
While certain analytical limitations can be overcome by using alternative methods or assays (e.g. fructosamine in patients with complex hemoglobinopathies), clinical conditions should be considered for accurate evaluation of glycemic level.
Medical conditions that decrease the life span, number of RBC or the quantity of hemoglobin (e.g. Fe-deficient anemia, hemolytic disease or conditions, splenomegaly, B12/B9 deficiency, renal disease, ineffective erythropoiesis, etc.) will lead to a decrease in glycation time. Such patients will have falsely low HbA1c, independent of the glucose concentration.
Medical conditions that increase the life span or number of RBC (e.g. increase production of RBC post erythropoietin administration in patients with CKD and inefficient erythropoiesis) will lead to an increase in glycation time. Therefore, patients will have falsely elevated HbA1c, independent of the glucose concentration.
Various other conditions (liver and biliary diseases with hyperbilirubinemia, alcohol addiction, pregnancy), supplements (e.g. increase Vit.C and E), medication (e.g. opiates used in pain management, via negatively modulating the process of glycation and endocrine glycemic control) can also interfere with effective glycation of HbA, independent of the concentration of plasma glucose and age, leading to deceiving low HbA1c.
Overall, accurate interpretation of the HbA1c lab results is extremely complex. Considering all the above, Hb A1c lab results are often overconfidently interpreted.
