The hemoglobin A1c (HbA1c) test, also known as a glycated hemoglobin test, is an assessment of long-term serum glucose levels that can be used to both diagnose type 2 diabetes and assess glycemic control in patients with diabetes.1-4 HbA1c test results are reported as a percentage and reflect the level of chronic exposure of red blood cells to glucose over the past 2-3 months.
In the bloodstream, ambient glucose undergoes nonenzymatic attachment to available amino groups on proteins of red blood cells, referred to as glycation.5 Hemoglobin that has attached glucose is referred to as glycated hemoglobin (GHb). HbA1c tests measure glucose attachment to a specific area on hemoglobin type A1, which is comprised of HbA1a, HbA1b, and HbA1c. Glycation of HbA1c has been found to be analogous to overall presence of glucose in the blood over the lifespan of the hemoglobin.6
Glycation of HbA1c occurs through a multi-step process in the presence of ambient glucose.5 First, glucose binds noncovalently and reversibly to the N-terminal valine (or select lysine residues) on the β-chain of hemoglobin. A Schiff base or aldimine then forms via intermolecular reaction of an electrophilic anomeric carbon and nucleophilic nitrogen, followed by an irreversible conformational change that forms the final Amadori intermediate and then the final glycosylated product.
The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) published standardization of HbA1c testing assays about two decades ago to ensure systematically consistent measurements.7 The HbA1c test is performed with a small blood sample, typically collected from the arm or by a finger prick. Over 30 laboratory HbA1c testing methods are available on the market for provider use. Ion exchange chromatography and capillary electrophoresis methods utilize charge differences in the glycated and non-glycated hemoglobin to calculate HbA1c.7 Immunoassay, enzymatic assay, and affinity chromatography methods differentiate glycated and non-glycated hemoglobin via their structural differences.
The HbA1c test is beneficial in a number of ways, including measuring relatively long-term glycemic control, not requiring fasting, having low day-to-day variability in patients with no hemoglobinopathies, having good preanalytical stability, and having assay standardization.1-3 Because HbA1c levels reflect chronic hyperglycemia and avoid the sensitive variability of blood glucose levels, it is considered optimal for monitoring diabetes over time.
There are some limitations to the HbA1c test, including generally being more expensive, less globally available compared to other forms of glucose testing, and the potential for inaccurate results in some patients.1,2 Several conditions can affect HbA1c test results, including altered erythrocyte turnover (e.g., anemia, iron status, splenectomy, blood loss, transfusion, hemolysis, glucose-6-phosphate dehydrogenase deficiency, erythropoietin), human immunodeficiency viruses (HIV), cirrhosis, renal failure, dialysis, or pregnancy. Race has also been identified as a potential confounder, with some studies describing higher hemoglobin glycation in patients of black African origin than white European origin.8 However, inaccuracy in test results is relatively uncommon. A 2020 retrospective study found that out of 12,279 tests, 0.9-2.1% were falsely elevated.9 Implementing a 60-day retesting protocol reduced this number to 0.2%.
Both the American Diabetes Association (ADA) and the World Health Organization (WHO) recommend HbA1c testing for the diagnosis of type 2 diabetes, with the threshold for diagnosis of ≥6.5%.1,2 There remains some debate on whether the HbA1c test should be used as a diagnostic tool for prediabetes. The ADA recommends HbA1c testing for the diagnosis of prediabetes with a positive diagnosis for results ranging from 5.7-6.4%.1 However, the WHO does not endorse the use of HbA1c levels as a diagnostic criteria for prediabetes.
Ultimately, the HbA1c test is considered a reliable measurement of glycemic control and is used regularly in the management of both patients with diabetes and prediabetes.
References
- 2. Diagnosis and Classification of Diabetes: Standards of Care in Diabetes-2024. Diabetes Care. Jan 1 2024;47(Suppl 1):S20-s42. doi:10.2337/dc24-S002
- HEARTS D: diagnosis and management of type 2 diabetes. 2020. 22 April 2020. https://iris.who.int/bitstream/handle/10665/331710/WHO-UCN-NCD-20.1-eng.pdf;sequence=1
- Inzucchi SE. Clinical practice. Diagnosis of diabetes. N Engl J Med. Aug 9 2012;367(6):542-50. doi:10.1056/NEJMcp1103643
- Sacks DB, Arnold M, Bakris GL, et al. Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus. Diabetes Care. 2023;46(10):e151-e199. doi:10.2337/dci23-0036
- Clark SLD, Santin AE, Bryant PA, Holman RW, Rodnick KJ. The initial noncovalent binding of glucose to human hemoglobin in nonenzymatic glycation. Glycobiology. 2013;23(11):1250-1259. doi:10.1093/glycob/cwt061
- Khan HA, Ola MS, Alhomida AS, Sobki SH, Khan SA. Evaluation of HbA1c criteria for diagnosis of diabetes mellitus: a retrospective study of 12 785 type 2 Saudi male patients. Endocr Res. 2014;39(2):61-5. doi:10.3109/07435800.2013.828740
- Jeppsson JO, Kobold U, Barr J, et al. Approved IFCC reference method for the measurement of HbA1c in human blood. Clin Chem Lab Med. Jan 2002;40(1):78-89. doi:10.1515/cclm.2002.016
- Unwin N, Howitt C, Rose AM, Samuels TA, Hennis AJ, Hambleton IR. Prevalence and phenotype of diabetes and prediabetes using fasting glucose vs HbA1c in a Caribbean population. Journal of global health. Dec 2017;7(2):020407. doi:10.7189/jogh.07.020407
- Mrazek C, Stechemesser L, Haschke-Becher E, et al. Reducing the probability of falsely elevated HbA1c results in diabetic patients by applying automated and educative HbA1c re-testing intervals. Clin Biochem. Jun 2020;80:14-18. doi:10.1016/j.clinbiochem.2020.03.014
