Hemoglobin A1c (A1c) is an essential component to both the diagnosis and management of patients with diabetes mellitus. A1c is a specific glycated hemoglobin (Hb) that is modified at the N-terminal valine residue of each ß-chain of Hb A. A1c levels reflect not only average blood glucose concentrations during the previous 8 to 12 weeks but also long-term glycemic control. The landmark Diabetes Control and Complications Trial showed that A1c is directly related to risks for diabetic complications, and as a result, clinical guidelines recommend specific treatment goals related to A1c levels.

Measuring A1c is an integral component of the standard of care for diabetic patients, and there are a variety of methods to do so. A1c methods fall into two broad categories: those that use molecular charge (ion-exchange high performance liquid chromatography [HPLC], and electrophoresis) and those that use molecular structure (immunoassay and affinity chromatography). Nearly all A1c measurements in the U.S. are performed with these methods, though point-of-care (POC) devices also are used widely.

A clinical laboratory’s choice of A1c methods depends on factors such as cost, patient population, existing instrumentation, performance, and available resources. When selecting an A1c method labs should first determine if the method is National Glycohemoglobin Standardization Program (NGSP)-certified—better than 99% in the U.S. are—and evaluate College of American Pathologists (CAP) data to assess how well the method actually performs. With any method, if A1c concentrations do not match the clinical picture, then biologic factors—such as altered erythrocyte lifespan—should be considered.

Additionally, laboratories should be aware of the limitations their method has with respect to Hb variants and communicate this with their clinical colleagues. Efforts should be made to identify Hb variants whenever possible. And if necessary, alternative methods, such as fructosamine, should be selected to monitor these patients with diabetes.

Hb Variant Detection Varies Across Methods

Cation-exchange HPLC separates glycated from non-glycated Hb components based on differences in their charges, as glycation of the N-terminal residue decreases its positive charge. Several fully automated systems are available for the clinical laboratory and analysis time is typically less than 5 minutes. According to a 2014 CAP survey, the coefficient of variation (CV) for this method was 1.6–2.7%. More common Hb variants can usually be detected by examining the chromatogram, but if the variant can’t be separated from Hb A or A1c, incorrect levels will be reported. Even though ion-exchange HPLC allows for Hb variant detection and has good precision, implementing this platform into the clinical laboratory can be demanding in terms of necessary operator skills and the financial burden of equipment purchase.

Immunoassays exploit structural variations and use antibodies that target N-terminal glycated amino acids on the ß chain to quantify A1c. Immunoassays are commercially available and, according to the 2014 CAP survey, have variable CVs between 1.6–6.1%. Interferences from Hb variants depend on where the antibody is targeted and whether a patient’s mutation is in the first few amino acids of the ß chain.

Patients with Hb F levels above 10% will have a falsely low A1c because the gamma chain of Hb F shares only four of the 10 first amino acids with the ß chain of Hb A, and therefore has little to no immunoreactivity with most antibodies. On the plus side, immunoassays are affordable, easy to operate, and also easy to add to existing platforms in the laboratory. However, labs need to look carefully at the antibodies any particular assay targets and the patient population served to ensure the method will yield reliable results, or be prepared to investigate when test results don’t match the patient’s clinical picture.

Boronate affinity chromatography also relies on structural variations to detect and quantify Hb with attached glucose residues. m-amino­phenylboronic acid reacts with the glucose bound to Hb to selectively hold the glycated hemoglobin (GHb) on the column. Commercial assays are available, and according to the CAP 2014 survey this technique has reasonably good precision with a reported CV of 2.1–3.1%. This method has less interference from common Hb variants, but like immunoassays, it cannot detect the presence of variants and is affected by elevated Hb F levels above 10–15%. This is thought to result from a lower glycation rate for Hb F compared with Hb A. Therefore, like HPLC, affinity chromatography benefits from good precision and less interference from Hb variants. However, the method may require additional equipment and technical staff that may be cost prohibitive.


POC devices that measure A1c provide clear benefit in certain clinical situations. For example, during an in-office visit with clinicians, immediate test results allow for immediate therapy decisions and potentially better adherence to therapy. In the past, POC instruments fell short of the accepted analytic performance and NGSP certification. However, in recent years new instruments have come on the market with improved analytic performance. Even so, the American Diabetes Association concluded in 2014 that while POC A1c assays may be NGSP-certified they should not be used for diagnostic purposes.

Although a powerful clinical tool, A1c analysis has limitations. For example, A1c is altered by red blood cell lifespan. Hb variants may also interfere with A1c measurement, independent of their effects on erythrocyte survival. Pathologic variants of Hb such as Hb S or Hb E, elevated Hb F, and chemically modified derivatives of Hb (carbamylated Hb) seen in patients with renal failure can affect the accuracy of measurements from some methods. However, less information is available on interferences from patients with common Hb variants present in the homozygous state. The NGSP provides details on the effect of common Hb variants on a number of different methods in clinical use and is a useful resource when choosing an A1c method.

Timothy Hanley, MD, PhD, is a pathology resident and Heather Signorelli, DO, is a clinical chemistry fellow at ARUP Laboratories in Salt Lake City, Utah.

+Email: timothy.hanley@aruplab.com

+Email: heather.signorelli@aruplab.com