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StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-.

StatPearls [Internet].

Show detailsTreasure Island (FL): StatPearls Publishing; 2025 Jan-.Search term Hemoglobin A1C

Emily Eyth; Muhammad Zubair; Roopa Naik.

Author Information and Affiliations

Authors

Emily Eyth1; Muhammad Zubair2; Roopa Naik3.

Affiliations

1 University of South Florida2 Pakistan Kidney & Liver Institute and Research Centre - PKLI3 Centra Southside Community Hospital

Last Update: June 2, 2025.

Introduction

The hemoglobin A1c test—also known as glycated hemoglobin, glycosylated hemoglobin, HbA1c, or simply A1c—is used to measure an individual's glucose control levels. The test shows average blood sugar levels over the past 90 days, expressed as a percentage. In addition, it can be used to diagnose diabetes mellitus.[1] Hemoglobin is a protein found exclusively in red blood cells, giving blood its bright red color. The primary role of hemoglobin is to carry oxygen from the lungs to all the cells in the body. Hemoglobin becomes glycated or coated with glucose from the bloodstream. As blood glucose levels increase, more glucose attaches to the hemoglobin protein, resulting in a higher A1c value.[2] Since red blood cells have an average lifespan of about 3 months, the A1c test measures hemoglobin levels in the bloodstream over this period, making it a reliable indicator of blood sugar control.

Etiology and Epidemiology

The Diabetes Control and Complications Trial (DCCT) was a landmark study that provided extensive data on A1c and its correlation to blood glucose levels, establishing specific treatments to target A1c goals. Following the completion of the trial, the National Glycohemoglobin Standardization Program (NGSP) was formed to define a standardized assay for A1c measurement across laboratories. The DCCT trial reported that a higher mean A1c level was the dominant predictor of diabetic retinopathy progression.[3][4] Tighter glucose control, indicated by HbA1c levels at or below 7%, was correlated with a 35 to 76% decrease in microvascular complications, such as retinopathy, nephropathy, and neuropathy, in patients with type 1 diabetes. In addition to predicting the progression of microvascular complications through A1c levels, the DCCT extension into the Epidemiology of Diabetes Interventions and Complications study showed long-term benefits in cardiovascular risk and mortality for patients with lower HbA1c levels.[5][6]

Pathophysiology

Regular A1c testing is essential for individuals with diabetes mellitus to ensure their average blood glucose levels are within the target range. The American Diabetes Association (ADA) recommends that individuals with diabetes mellitus who have stable blood sugar control and are meeting treatment goals should have their HbA1c (A1C) checked at least twice a year.[2] For individuals whose therapy has changed or who are not meeting glycemic targets, testing should be performed every 3 months. This approach helps optimize diabetes control and reduce the risk of complications.[7]

Specimen Requirements and Procedure

The HbA1c test can be performed as a point-of-care (POC) test, a STAT test, or by sending a sample to a laboratory. The POC test uses a STAT analyzer to measure A1c from a capillary fingerstick. The laboratory test uses a teaspoon of blood drawn from a venous sample into a K2 EDTA (lavender top) tube. The sample is processed as whole blood. Patients do not have to fast for the HbA1c test, as it reflects long-term blood sugar control rather than immediate glucose levels.[2] 

Diagnostic Tests

The venous sample A1c test may be used as a diagnostic tool in clinical practice when determining diabetes risk or onset. Due to the variability of capillary POC testing, any A1c test conducted using a capillary sample should be confirmed with a venous sample before diagnosing. An HbA1c value below 5.7% is considered normal or non-diabetic. A value between 5.7% and 6.4% indicates prediabetes mellitus, whereas a 6.5% or higher level is diagnostic for diabetes mellitus. Tests should be sent to a laboratory certified by the NGSP to ensure standardized results.[8][9]

Testing Procedures

Numerous analytical techniques have been developed to quantify HbA1c, each offering specific advantages in precision and applicability. To ensure global consistency, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) established a reference system for HbA1c measurement. This system uses primary reference materials composed of purified HbA1c and HbA0, with 2 proposed reference methods—electrospray ionization mass spectrometry and capillary electrophoresis.[10]

These reference methods target the glycated N-terminal valine of the hemoglobin β-chain. The process involves enzymatic digestion with endoproteinase Glu-C to release the N-terminal hexapeptide, followed by separation and quantification of glycated and non-glycated hexapeptides.[11] HbA1c is expressed as the ratio of glycated to total hexapeptides. A network of IFCC-designated reference laboratories maintains and supports this standardized framework, ensuring its reliability for assay calibration.[12]

Comparative analyses of the IFCC system with the NGSP and other regional standards demonstrate robust correlation, enabling manufacturers to align their assays with the IFCC reference method.[13] However, HbA1c values derived from IFCC methods are typically 1.5% to 2% lower than NGSP values due to differences in the glycated fractions measured. The IFCC method is not intended for routine clinical use but is a calibration standard for commercial assays. For clinical testing, NGSP-certified methods are recommended.[14]

In laboratory settings, high-performance liquid chromatography (HPLC) using cation-exchange chromatography remains a cornerstone technique for HbA1c measurement. This method effectively removes labile intermediates that may interfere with alternative approaches, such as immunoassays or affinity chromatography. Fully automated HPLC systems are widely available, requiring minimal sample volumes (approximately 5 μL of whole blood). Although venous blood is standard, capillary blood from a finger prick is also acceptable. Samples are diluted with a borate-containing hemolysis reagent and incubated at 37 °C for 30 minutes to eliminate labile Schiff bases before analysis.[15][16]

Immunoassay-based methods are also prevalent, using monoclonal antibodies that specifically bind to the Amadori product (ketoamine linkage) and distinct amino acid sequences at the N-terminal of the hemoglobin β-chain. A widely used format, latex agglutination inhibition, involves a synthetic polymer bearing HbA1c epitopes that competes with patient HbA1c for binding to antibody-coated latex beads. This competition reduces agglutination, which is quantifiable as decreased absorbance. Enzyme immunoassays using analogous antibodies exhibit acceptable precision and are typically calibrated to HPLC-derived values. These assays are highly specific, excluding labile intermediates; other glycated hemoglobin, such as HbA1a and HbA1b; and common hemoglobin variants, such as HbF, HbA2, HbS, and carbamylated hemoglobin.[17][18]

Recent advancements have introduced enzymatic assays using fructosyl peptide oxidase for HbA1c quantification, alongside automated liquid-flow capillary electrophoresis systems, which provide viable alternatives for routine clinical testing.[19]

POC devices are increasingly used for HbA1c measurement in clinical settings. However, the diversity of available POC systems complicates selection, as comparative performance data are limited. Notably, POC measurements typically yield values approximately 0.5% lower than laboratory-based assays and may exhibit reduced accuracy compared to venous blood samples analyzed in a laboratory. Inter-laboratory variability in HbA1c results can reach 0.5%, underscoring the importance of standardized methodologies to ensure reliable clinical outcomes.[20][21]

Interfering Factors

Several conditions can interfere with the accuracy of HbA1c testing. Individuals with sickle cell anemia, thalassemia, anemia, kidney failure, liver disease, or those receiving blood transfusions can experience altered results due to the longevity of the red blood cell. HbA1c measurement in these patients must be interpreted cautiously and confirmed with plasma glucose samples to diagnose diabetes mellitus.[22] A falsely low HbA1c value can result from several conditions, including high altitude, pregnancy, hemorrhage, blood transfusion, erythropoietin administration, iron supplementation, hemolytic anemia, chronic kidney failure, liver cirrhosis, alcoholism, sickle cell anemia, and spherocytosis. Vitamin C supplementation can increase or decrease HbA1c levels depending on the method used.[23][24]

Conversely, a falsely high HbA1c value can be due to insufficient iron in the blood. This condition can result from iron deficiency anemia, infection-induced or tumor-induced anemia. In addition, hemoglobinopathies such as thalassemia and B12 deficiency can cause a falsely high HbA1c value.[25][26] Other causes of falsely high HbA1c levels include hypertriglyceridemia, organ transplantation, and hyperglycation in certain ethnic groups. Medications such as immunosuppressants and protease inhibitors can sometimes lead to a falsely high HbA1c value.[22][27][28]

Results, Reporting, and Critical Findings

The HbA1c percentage reflects the average glucose level in a patient's body over the past 90 days.[29][30][31]

Table

Table. Relationship Between HbA1c and Average Glucose Levels.

Clinical Significance

HbA1c is a well-established marker that reflects average blood glucose levels over the preceding 3 months and serves as a reliable indicator of long-term glycemic control.[2] HbA1c is widely used not only for monitoring diabetes mellitus but also as a diagnostic tool and an alternative to glucose testing for screening diabetes mellitus. Glycated hemoglobin has been firmly recognized as a predictor of the risk of developing microvascular complications, such as retinopathy and nephropathy, in patients with diabetes mellitus.[32][33] Notably, the absolute risks of these complications are directly proportional to the mean HbA1c concentration. Studies have demonstrated that the risk of diabetic retinopathy increases continuously with rising HbA1c levels, and a single HbA1c measurement can predict the progression of retinopathy even 4 years later.[2][34]

Quality Control and Lab Safety

The laboratory must develop, document, implement, and maintain a comprehensive quality management system to ensure all processes adhere to established quality standards and consistently deliver reliable results.[35] For HbA1c testing, both internal and external quality control are vital elements of this system, directly contributing to the accuracy and consistency of reported outcomes.[36]

Internal quality control focuses on monitoring the analytical phase of the testing process. According to Clinical Laboratory Improvement Amendments guidelines, a minimum of 2 quality control levels must be performed every 24 hours for routine laboratory assays. Although manufacturers provide control ranges, laboratories are encouraged to define them based on internal data and relevant guidelines to optimize performance.[37] Quality control results should be charted using Levey-Jennings plots, with appropriate Westgard rules applied. If any control rule is violated, patient testing must be halted immediately, appropriate corrective actions implemented, and testing resumed only after control results return to acceptable limits.[38]

External quality assessment, or proficiency testing, offers an objective evaluation of laboratory performance. Participating laboratories receive blinded samples from an external organization, submit their results, and have them statistically analyzed and compared against peer groups. For HbA1c, the College of American Pathologists provides an accuracy-based external quality assurance program that promotes interlaboratory consistency and helps identify areas needing improvement.[39][36][40]

Given that HbA1c exhibits low within-subject biological variation, typically <2%, the ADA recommends that intralaboratory imprecision maintain a coefficient of variation (CV) below 2%, and interlaboratory CV should not exceed 3.5%.[41] Meeting these precision standards guarantees the reliability and clinical significance of test results.

Laboratory safety is equally critical to overall testing quality. All personnel must follow infection prevention protocols, including proper use of personal protective equipment and safe handling of biological specimens. Maintaining a safe and compliant laboratory environment helps ensure consistent performance while minimizing risks of errors or exposure.[42]

Enhancing Healthcare Team Outcomes

All clinicians caring for patients with diabetes mellitus need a thorough understanding of HbA1c and its clinical significance. In general, HbA1c reflects the average glucose concentration over 3 months. HbA1c is often used as an outcome measure to determine whether an intervention in a population is successful by demonstrating a decrease in HbA1c by a specific percentage. There is a movement within the medical community to shift away from using HbA1c as an exclusive standard of care test to assess patient response to treatment. The newest proposed methods include the estimated average glucose and the glucose time in range.[43] These methods use data obtained from continuous glucose monitors that record blood glucose levels around the clock. In addition, these methods can offer healthcare professionals a more accurate picture of the blood sugar average and fluctuations. However, these methods are not available to all patients on a widespread basis.

According to ADA guidelines, HbA1c levels should be measured 2 times annually in stable patients and at least 4 times in patients with glucose fluctuations or those who have changed their diabetes treatment. HbA1c is one of the preferred diabetes diagnostic tests today. The blood draw can occur anytime, and no special handling requirements exist. However, to ensure that the A1c value is correct, clinicians must be aware of the causes of false-positive and false-negative results.

As many patients with diabetes mellitus are treated in outpatient settings, diabetes care nurses must understand HbA1c results and know when to refer patients to an endocrinologist for further evaluation. Pharmacists must also fully understand and interpret this test, as they are involved in glycemic management, medication, agent selection, dosing, and monitoring. The nurse and pharmacist must inform the treating clinician regarding any changes in HbA1c and verify patient medication compliance. HbA1c is a valuable tool for managing diabetes mellitus and other glycemic control disorders, but its effectiveness is maximized within an interprofessional healthcare team environment.

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References

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Disclosure: Emily Eyth declares no relevant financial relationships with ineligible companies.

Disclosure: Muhammad Zubair declares no relevant financial relationships with ineligible companies.

Disclosure: Roopa Naik declares no relevant financial relationships with ineligible companies.

Copyright © 2025, StatPearls Publishing LLC.

This book is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ), which permits others to distribute the work, provided that the article is not altered or used commercially. You are not required to obtain permission to distribute this article, provided that you credit the author and journal.

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• Cite this PageEyth E, Zubair M, Naik R. Hemoglobin A1C. [Updated 2025 Jun 2]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-.

In this Page

• Introduction
• Etiology and Epidemiology
• Pathophysiology
• Specimen Requirements and Procedure
• Diagnostic Tests
• Testing Procedures
• Interfering Factors
• Results, Reporting, and Critical Findings
• Clinical Significance
• Quality Control and Lab Safety
• Enhancing Healthcare Team Outcomes
• Review Questions
• References

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• PubMedLinks to PubMed

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