Navigating hemoglobin A1C measurements for diabetes care

Diabetes mellitus, a chronic metabolic disorder characterized by persistent hyperglycemia, affects millions of people worldwide and poses significant health and economic challenges. An accurate diagnosis plays a crucial role in helping patients receive treatment as soon as possible to prevent complications. Traditional testing of patients’ glycemic status involves either measuring their blood glucose levels or assessing the amount of hemoglobin A1c (HbA1c) in their blood.

Assessing the relative concentration of HbA1c offers several advantages over direct glucose measurement. Most notably, it correlates effectively with the average concentration of serum glucose over a period of 2−3 months (1, 2), whereas glucose evaluations can provide insight only into a patient’s glucose status at the time of blood collection. For that reason, the American Diabetes Association (ADA) recommends the use of HbA1c as an indicator of long-term glycemic control in patients with diabetes mellitus, as well as for the screening and diagnosis of the disease, with a cutoff value of 6.5% (1) (See Table 1).

Given the importance of HbA1c in managing diabetes, it’s important for clinical lab professionals and healthcare providers to understand how results can be influenced by various factors, including analysis methods, clinical conditions, and other variables. Selecting the best approach requires careful assessment of each patient’s needs.

Table 1. Comparison of glucose and HbA1c testing modality, advantages and limitations.

Glucose evaluation	HbA1c evaluation
Fasting	No fasting necessary
Multiple draws (e.g., oral glucose tolerance test)	One draw
Reflects immediate variant interferences	Hb variants can interfere
No hemoglobin variant interferences	Hb variants can interfere
Standardized and reference methods established	In process of standardization A complex International Federation of Clinical Chemistry and Laboratory Medicine reference method has been developed

About HbA1c

HbA1c is produced by the nonenzymatic addition of a glucose molecule to the easily accessible N-terminal valine residue on the β chain of the hemoglobin A (HbA) molecule. Because no enzymatic limitation exists, the production of HbA1c reflects the exposure of HbA to the glucose level in the cellular environment in a directly proportional relationship. The higher the glucose level, the higher the HbA1c. However, stabilization (Amadori rearrangement) of hemoglobin’s glycated structure (such as reversable Schiff base, also known as labile-HbA1c) requires a continuous and lengthy exposure of HbA to glucose (stable-HbA1c with clinical significance) (3). Therefore, HbA1c correlates with the mean level of blood glucose over the life span of erythrocytes and HbA, which is 106±21 days (4, 5). Nevertheless, depending on the method, the labile-HbA1c may or may not be included in the measurement and calculation of stable-HbA1c.

Because the process of glycation is not enzymatically mediated but based on the biochemical properties of the -NH groups of amino acids and the aldehyde group of glucose and how they react with each other, there is no site-specific attachment of glucose to HbA. As such, glycation, the attachment of glucose residues at amino groups, can occur at any amino group or amino acid that is accessible to glucose. Although N-terminal valine residue on the β chain of HbA is the most accessible position in the HbA molecule, 85% of total glycated HbA is represented by HbA1c. However, additional hemoglobin glycated species, such as HbA1a1, HbA1a2, and HbA1b, also can be formed. Together, they are defined as the total glycated HbA. This is what the charge-based methods and electrophoresis actually evaluate.

Glycation of the N-terminal residue changes the structure and decreases the positive charge of HbA, allowing separation and quantification of glycated versus nonglycated HbA in clinical laboratories (6).

Types of HbA1c analysis

Methods of HbA1c analysis fit into two broad categories: Methods based on the separation and quantification of the glycated hemoglobin due to its different molecular charge in relationship with the nonglycated hemoglobin, and methods based on the identification of structural changes of the hemoglobin post glycation and quantification. The former category includes capillary electrophoresis-high performance liquid chromatography (CE-HPLC, e.g., Bio-Rad) and electrophoresis (e.g., Sebia Capillaries). The latter includes immunoassays (various manufacturers), boronate affinity chromatography (Trinity Biotech), and mass spectrometry (7).

CE-HPLC and electrophoresis assays

This method is able to quantitate HbA1c by separating it from HbA because glycation of the N-terminal valine decreases the positive charge. However, charge-based methods are susceptible to interference from nonglucose adducts such as carbamylation in uremia and acetylation, and structural changes from Hb variants that alter net charge. That affects the migration profile with electrophoresis or retention time with CE-HPLC of hemoglobin variants, which may migrate or coelute concurrently with HbA1c or HbA, causing incorrect interpretation. Currently, more than 1,000 human hemoglobin variants (alpha, beta, gamma, or delta variants) have been described. They result from point mutations that cause amino acid substitutions. Depending on their charge, these variants can lead to false increases or decreases in HbA1c (7).

Although these methods are not affected by most common Hb traits, such as Hb AS (the sickle cell trait) and Hb AC, they are not completely excluded from interference due to certain hemoglobin variants (11). Proper resolution and peak identification of HbA1c, HbA, and interfering variants is critical for accurate quantification (e.g., %HbA1c=AUC of HbA1c / AUC of total Hb) (7, 10). However, labile-HbA1c can interfere with HbA1c measurements and often requires pretreatment.

Immunoassays

This form of testing uses antibodies that target N-terminal glycated amino acids (generally the first four monoacids) on the β chain to quantify HbA1c. Labs calculate the HbA1c percentage based on the HbA1c and total Hb concentrations (11, 12, 13). Thus, any factor that prevents glycation or any amino acid substitution in the β-chain N-terminal epitope may hinder antibody binding, leading to falsely low or undetectable HbA1c values. This may occur, for example, in patients with hemoglobin variants such as HbS, which is present in people with sickle cell disease, and HbC, an inherited mutation.

Depending on the specificity (antibody recognition), glycated species resulting from hemoglobin variants may or may not be included in the measurements. Additionally, patients with high levels of fetal hemoglobin (HbF >10%), such as sickle-cell patients treated with hydroxyurea or individuals with a genetic condition in which they continue to produce HbF into adulthood, are prone to falsely low HbA1c value by immunoassay, since HbF lacks the β chain targeted by most assays. Although HbF is included in the calculation of the %HbA1c (%HbA1c=HbA1c/total Hb), HbF β chains share limited homology with the β-chain N-terminus, resulting in poor cross-reactivity with antibodies in most immunoassays (14). These assays were never harmonized and thus vary in sensitivity to hemoglobin variants, which leads to discrepancies across platforms.

Boronate affinity chromatography (BAC)

In this method, boronic acid reacts with cis-diol groups created by glycation, thereby allowing glycohemoglobins such as HbA1c to be very specifically separated from HbA (2). Due to the design of the assay, BAC is virtually free of interferences from hemoglobin variants. However, elevated HbF (>20%) can still lead to spurious results for the reasons mentioned above. Further, rare Hb variants with excessive glycation, such as Hb Himeji, also can interfere with boronate affinity chromatography (15). Still, BAC (Trinity Biotech) remains the method with the highest specificity for HbA1c currently on the market. This test specifically measures HbA1c and does not include other hemoglobin glycated species.

Thanks to its high specificity, BAC results correlate better than any other method with results obtained using the mass spectrometry-based reference method from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) mentioned below (8). It should also be noted that Abbott developed a point-of-care testing (POCT) instrument for robust evaluation of HbA1c that is based on this method (the Abbott Afinion HbA1 assay).

Although virtually no analytical interference affects HbA1c evaluation in patients with common hemoglobin variant traits regardless of the method, this is not the case in patients who have both β-globin alleles affected by mutation. This includes homozygous mutations such as Hb SS-sickle cell disease and Hb CC-hemoglobin C disease, or compound heterozygous mutations, such as Hb SC. Although the process of hemoglobin glycation still occurs, leading to formation of glycated variant hemoglobins, these patients do not have HbA and therefore do not produce HbA1c.

Mass spectrometry

The IFCC developed a mass-spectrometry–based technique for measuring HbA1c. Because of its higher specificity for evaluating the glycated N-terminal valine of the HbA β chain, the IFCC approved mass spectrometry as the international reference method for evaluating HbA1c. This approach involves a two-step process. First, the hemoglobin (glycated and nonglycated) is digested by the endoproteinase Glu-C to shorter polypeptides. Second, the glycated and non-glycated N-terminal hexapeptides of the β chain are separated and quantitated either by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization or by a two-dimensional approach using HPLC and capillary electrophoresis with UV-detection.

Both principles give identical results, and HbA1c is measured as the ratio between the glycated and nonglycated hexapeptides. The calibrators of this method consist of a mixture of highly purified HbA1c and HbA0 and represent an important step toward standardization of HbA1c testing. Precision and accuracy were high for this method, which was virtually free of any analytical interference. Although this method is considered the basis of the uniform standardization of HbA1c routine assays worldwide, it is not routinely used in clinical laboratories due to the complexity of the analytical steps involved, the prohibitive cost of a mass spectrometer, and the complicated nature of its installation and operation (6).

Point-of-care testing

Most of the POCT instruments used for evaluating HbA1c are based on immunoassay and are thus subject to the limitations of that technique, as well as significant performance variability due to reagents’ lot-to-lot variability (17). Further, the Food and Drug Administration (FDA) has authorized most POCT HbA1c instruments for monitoring HbA1c levels in patients with known diabetes and not for diagnostic purposes.

However, recently, the Afinion 2 HbA1c Dx cartridges received FDA-approval as a moderate complexity tool for diagnosing diabetes mellitus and helping to identify patients at high risk for developing it.

Spectroenzymatic method

A spectroenzymatic method for HbA1c also was developed recently. In this method, the hemoglobin in the sample is first oxidated using sodium nitrite, which results in the formation of methemoglobin. Measurements at 505/800 nm are used to determine the concentration of total hemoglobin. After this step, protease cleaves the connection between leucine and histidine at the N-terminal end of the β-chain, which leads to the formation of a glycated dipeptide fragment. This glycated dipeptide then undergoes a reaction with fructosyl peptide oxidase, resulting in the production of hydrogen peroxide. Subsequently, under the action of peroxidase, hydrogen peroxide reacts with a specific die. The concentration of HbA1c in the sample can be measured through spectrophotometric evaluation of the resulting complex. The HbA1c content and its percentage in total hemoglobin is then calculated by measuring the absorbance at 660/800 nm.

Currently, this method is not used routinely for HbA1c lab testing. However, one study has shown that it produces results with comparable accuracy to those obtained using the IFCC method and that it also demonstrates limited susceptibility to interferences (18). Because of its demonstrated high level of specificity and accuracy, it is possible that it might become a common method in the future.

Variations in testing methods

Several factors influence the selection of HbA1c testing methods in clinical laboratories, including cost, patient demographics, existing equipment, performance characteristics, and available resources. There is an intense effort to standardize HbA1c testing. About 99% of U.S. clinical laboratories use methods certified by the National Glycohemoglobin Standardization Program (NGSP), which aims to align HbA1c results with those of the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study.

These landmark studies established clear connections between HbA1c levels and outcomes in patients with diabetes. The NGSP Certified Network Laboratories employ methods that are calibrated and traceable to those used in the DCCT. By comparing results with the NGSP network, both manufacturers and clinical laboratories can ensure their glycated hemoglobin measurements align with DCCT standards, increasing the likelihood of meeting the College of American Pathologists’ (CAP) survey requirements.

The wide range of methods clearly contributes to significant analytical variability. The availability of numerous FDA-approved HbA1c testing platforms on the market further complicates this issue. Currently, according to ADA, the precision goals for HbA1c measurement are to have a coefficient of variation (CV) of <1.5% for intralaboratory tests and <2.5% for interlaboratory measures (using at least two control samples with different HbA1c levels), and ideally no measurable bias (19, 20).

According to the most recent CAP survey of HbA1c challenges released in January, 29 methods from over 1,000 participating laboratories achieved this criterion on proficiency testing, representing most of the available products on the market. Some methods, such as ARKRAY Adams HA-8190V, had a CV<1.5%. Eight other methods performed at a bias <2%, including the Biorad D-100, Sebia Cappilarys, Siemens Atellica, Tosoh, Trinity Biothec Premier, and all three Abbott methods. A few methods did not meet the ADA criteria, performing at a CV > 2.5% or even >3% (Roche cobas, Siemens DCA Vintage, Vitros Chemistry Systems, and Beckman AU Systems). Like other CAP challenges, the survey evaluated the results against the NGSP and IFCC reference method target and considered acceptable results within ±6% of the target value.

Biological and clinical factors

In addition to method-specific analytical issues, correct evaluation of HbA1c also depends on biological and clinical factors (7). Conditions that shorten the lifespan of red blood cells (RBCs), such as hemolysis and anemia, result in lower HbA1c levels because of insufficient exposure time to glucose, regardless of glycemia. Similarly, decreases in the number of RBC or the quantity of hemoglobin will lead to a decrease in glycation time. This can occur with numerous conditions, including iron-deficiency anemia, hemolytic disease, splenomegaly, B12/B9 deficiency, renal disease, and ineffective erythropoiesis. Patients with these conditions will have falsely low HbA1c independent of the glucose concentration (20, 21).

Medical conditions that increase the life span or number of RBCs, including chronic kidney disease, inefficient erythropoiesis, or repeated transfusions, will lead to an increase in glycation time. Therefore, patients will have falsely elevated HbA1c independent of the glucose concentration (20, 21).

Although certain analytical limitations can be overcome by using alternative methods or assays — for example, fructosamine in patients with complex hemoglobinopathies — clinical conditions should be considered for accurate evaluation of glycemic level (7, 21, 22).

Many other factors can interfere with effective glycation of HbA independent of the concentration of plasma glucose and age. These include various conditions such as liver and biliary diseases with hyperbilirubinemia, alcohol addiction, and pregnancy; supplements such as vitamins C and E; and medication such as opiates used in pain management, which negatively modulate the process of glycation and endocrine glycemic control (7).

Conclusion

Although HbA1c measurement is a powerful tool in diabetes management, the accurate interpretation of results is extremely complex. Without assessing the full context, results could be interpreted overconfidently. Before selecting a tool, healthcare professionals should carefully consider analytical, pathophysiological, and other factors that could affect the accuracy of measurement for every patient.

Alina Gabriela Sofronescu, PhD, NRCC, FADLM, is an associate professor in the department of pathology at the Atrium Wake Forest Baptist Medical Center in Winston-Salem, North Carolina. +Email: [email protected]

Read the full September-October issue of CLN here.

References

1. American Diabetes Association. Standards of medical care in diabetes - 2010. Diabetes Care 2010;33:S11–61.
2. Gillett MJ. International Expert Committee Report on the Role of the A1C Assay in the Diagnosis of Diabetes. Clin Biochem Rev 2009;30:197–200.
3. Acharya AS, Roy RP, Dorai B. Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin A: preferential inhibition of glycation by nucleophiles at sites of low isomerization potential. J Protein Chem 1991;10:345-58.
4. Cohen RM, Franco RS, Khera PK, et al. Red cell life span heterogeneity in hematologically normal people is sufficient to alter HbA1c. Blood 2008; doi: 10.1182/blood-2008-04-154112.
5. Khera PK, Smith EP, Lindsell CJ, et al. Use of an oral stable isotope label to confirm variation in red blood cell mean age that influences HbA1c interpretation. Am J Hematol 2015; doi: 10.1002/ajh.23866.
6. Sacks DB. A1C versus glucose testing: A comparison. Diabetes Care 2011;34:518–23.
7. Sofronescu AG, Williams LM, Andrews DM, et al. Unexpected hemoglobin A1c results. Clin Chem 2011;57:153-6.
8. Weykamp CW, Penders TJ, Muskiet FA, et al. Influence of hemoglobin variants and derivatives on glycohemoglobin determinations, as investigated by 102 laboratories using 16 methods. Clin Chem 1993;39:1717-23.
9. Allen DW, Schroeder WA, Balog J. Observations on the chromatographic heterogeneity of normal adult and fetal human hemoglobin: A study of the effects of crystallization and chromatography on the heterogeneity and isoleucine content. J Am Chem Soc 1958;80:1628–34.
10. Bry L, Chen PC, Sacks DB. Effects of hemoglobin variants and chemically modified derivatives on assays for glycohemoglobin. Clin Chem 2001;47:153–63.
11. Little RR, Rohlfing CL, Hanson SE, et al. The effect of increased fetal hemoglobin on 7 common HbA1c assay methods. Clin Chem 2012;58:945-7.
12. Roberts WL, Frank EL, Papadea C, et al. Effects of nine hemoglobin variants on five glycohemoglobin methods. Clin Chem 2000;46:560-76.
13. Schnedl WJ, Liebminger A, Roller RE, et al. Hemoglobin variants and determination of glycated hemoglobin (HbA1c). Diabetes Metab Res Rev 2001;17:94-8.
14. Chen Z, Shao L, Zhiang M, et al. Interpretation of HbA1c lies at the intersection of analytical methodology, clinical biochemistry, and hematology (Review). Exp Ther Med 2022; doi: 10.3892/etm.2022.11643.
15. Koga M, Inada S, Shimizu S, et al. Aldimine formation reaction, the first step of the Maillard early-phase reaction, might be enhanced in variant hemoglobin, Hb Himeji. Ann Clin Lab Sci 2015;45:643-9.
16. Jeppsson JO, Kobald U, Barr J et al. International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Approved IFCC reference method for the measurement of HbA1c in human blood. Clin Chem Lab Med 2002; doi: 10.1515/CCLM.2002.016.
17. Korpi-Steiner N, Cotten SW, Little RR, et al. Discordant point-of-care and laboratory hemoglobin A1c concentrations in ambulatory settings. J Appl Lab Med 2025; doi: 10.1093/jalm/jfae153.18. Li M, Wu X, Xie W, et al. Analytical performance evaluation of the Mindray enzymatic assay for hemoglobin A1c measurement. Sci Rep 2024; doi: 10.1038/s41598-024-63261-y.
18. Sacks DB. Measurement of hemoglobin A1c. Diabetes Care 2012;35:2674-80.
19. Sacks DB, Arnold M, Bakris GL, et al. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clin Chem 2023; doi: 10.1093/clinchem/hvad080.
20. Panzer S, Kronik G, Leckner K, et al. Glycosylated hemoglobins (GHb): An index of red cell survival. Blood 1982;59:1348-50.
21. Spencer DH, Grossman B, Scott MG. Red cell transfusion decreases hemoglobin A1c in patients with diabetes. Clin Chem 2011;57:344.