What is hemoglobin A1c (HbA1c)?

What is HbA1c?

HbA1c (hemoglobin A1c) is a stable glycated hemoglobin molecule formed by the nonenzymatic attachment of glucose to the N-terminal valine residue of the hemoglobin β-chain, reflecting average blood glucose levels over the preceding 2–3 months. 1

Biochemical Formation

• Glucose first forms a reversible Schiff base (aldamine) with the N-terminal valine on the β-chain of hemoglobin 1
• This aldamine then undergoes an Amadori rearrangement to form a stable ketoamine structure (N-1-deoxyfructosylhemoglobin), which is the actual HbA1c molecule 1
• The glycation process is nonenzymatic, continuous, and irreversible once the stable ketoamine is formed 1

Time Frame and Glucose Correlation

• HbA1c reflects glycemic exposure over approximately 2–3 months, corresponding to the average lifespan of red blood cells (approximately 120 days) 1
• The weighting is not uniform: approximately 50% of the HbA1c value reflects glucose levels from the most recent month, 25% from 30–60 days prior, and 25% from 60–120 days prior 2
• Each 1% increase in HbA1c corresponds to approximately a 35 mg/dL (2 mmol/L) increase in mean plasma glucose 3, 2

Clinical Applications

Diagnosis of Diabetes

• HbA1c ≥6.5% (≥48 mmol/mol) is diagnostic of diabetes when performed using an NGSP-certified method in an accredited laboratory 1
• HbA1c can serve as a supplementary diagnostic criterion, though confirmation with repeat testing or glucose-based criteria is recommended 1
• HbA1c values of 5.7%–6.4% (39–46 mmol/mol) define prediabetes or high risk for developing diabetes 1

Monitoring Glycemic Control

• HbA1c is the gold standard for assessing long-term glycemic control in diabetes management 1
• Testing should be performed every 3 months until glycemic targets are achieved, then every 6 months once stable control is maintained 1
• For patients whose therapy has changed or who are not meeting goals, quarterly testing is recommended 1, 4

Key Advantages Over Point Glucose Testing

• HbA1c is not affected by short-term fluctuations from diet, exercise, stress, or illness 1
• No fasting is required; blood can be drawn at any time of day 1
• HbA1c is a very stable chemical with minimal preanalytic variability 1
• Strong evidence links HbA1c levels to the risk of chronic microvascular complications (retinopathy, nephropathy, neuropathy) 1, 3, 5

Measurement Standardization

• HbA1c assays are standardized through two major programs: the National Glycohemoglobin Standardization Program (NGSP) in the United States and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference system 1
• Results can be reported as percentages (NGSP/DCCT-aligned) or as mmol/mol (IFCC units) 1
• Laboratories must use IFCC and/or NGSP-certified instruments to ensure accuracy and comparability of results 1

Measurement Methods

Charge-Based Methods

• Ion exchange high-performance liquid chromatography (HPLC) 1
• Capillary electrophoresis 1

Structure-Based Methods

• Boronate affinity HPLC 1
• Immunoassay techniques 1

Critical Limitations and Interfering Factors

Conditions That Falsely Lower HbA1c

• Any condition that shortens red blood cell lifespan will falsely decrease HbA1c 1, 3
• Hemolytic anemia (autoimmune, drug-induced, hereditary) 3, 2, 6
• Chronic liver disease with increased red cell turnover 1
• Splenomegaly 1
• Recent blood loss or transfusion 3, 2
• Pregnancy (due to increased red cell turnover) 1
• Administration of erythropoietin, iron, or vitamin B12 1
• Chronic kidney disease (due to reduced RBC lifespan and hemolysis) 3, 2

Conditions That Falsely Elevate HbA1c

• Any condition that prolongs red blood cell lifespan will falsely increase HbA1c 1, 3
• Iron deficiency anemia 1, 3, 2
• Vitamin B12 deficiency 1
• Aplastic anemia 3
• Splenectomy 1
• Carbamylation of hemoglobin in uremia 2

Hemoglobin Variants

• Hemoglobinopathies (HbS, HbC, HbE) can interfere with HbA1c measurements depending on the assay method used 3, 2
• In patients with hemoglobin variants or conditions affecting red cell turnover, only glucose-based criteria should be used for diagnosis and monitoring 3, 2

When HbA1c Is Unreliable

In the following situations, use only glucose-based testing (fasting plasma glucose or oral glucose tolerance test) for diagnosis and monitoring: 1, 3, 2

• Sickle cell disease or other hemoglobinopathies 1, 3
• Hemolytic anemia of any cause 3, 2, 6
• Pregnancy 1
• Recent significant blood loss or transfusion (within 2–3 months) 3, 2
• Advanced chronic kidney disease (eGFR <30 mL/min/1.73 m²) 2

Alternative Markers When HbA1c Is Unreliable

• Glycated albumin or fructosamine can be used when HbA1c is unreliable, as these markers reflect glycemia over 2–4 weeks and are independent of red cell turnover 3, 2
• However, these alternative markers lack the robust evidence linking them to long-term complications that HbA1c possesses 3
• Continuous glucose monitoring (CGM) is particularly useful when HbA1c values are discordant with clinical presentation or self-monitored glucose readings 3, 2

Common Clinical Pitfalls

• Never diagnose diabetes based on a single HbA1c without confirmation, especially when clinical presentation is discordant 3
• Point-of-care HbA1c devices should not be used for diagnostic purposes due to lack of standardization 3
• In diabetic patients with hemolytic anemia, HbA1c will be falsely low and is a very poor marker for glycemic control 6
• HbA1c cannot detect acute glycemic variability, hypoglycemia, or postprandial hyperglycemia—these require self-monitoring or CGM 3, 2