Three things laboratories need to know about HbA1c testing

It has been several decades since HbA1c testing made its debut in the arena of diabetes management. Since then, this breakthrough test has become a widely used and a highly effective complement to blood glucose testing for monitoring longer-term glucose levels in patients with diabetes and/or suspected diabetes. As the use of HbA1c testing has grown, so has the pool of clinical experience and evidence that drives best practices for quality patient care. This includes awareness of those conditions and circumstances that can compromise HbA1c testing’s utility. Attention to these factors helps assure laboratorians and clinicians that the results at the foundation of patient care decisions are accurate. To give adequate context to these influences, it is helpful first to understand the mounting global healthcare burden resulting from diabetes and the role of HbA1c testing in patient diagnosis and management.

Diabetes: A growing threat

Diabetes is a looming healthcare crisis. The International Diabetes Federation (IDF) reported in 2017 that the number of patients with diabetes stood at 425 million globally—or one in 11 people—and that half of those cases were undiagnosed.¹ The current figure is up from 108 million in 1980, as cited by the World Health Organization (WHO).² WHO reported that 8.5 percent of adults 18 years and older had diabetes in 2014 versus 4.7 percent in 1980.² This prevalent and life-threatening condition was the direct cause of 1.6 million deaths in 2016, making it the seventh leading cause of death in the world that same year.² The incidence of diabetes is expected to grow. By 2045, IDF estimates state that there will be 629 million people with diabetes in the world—a 48 percent increase from current levels.¹

HbA1c testing

The development of HbA1c assays was a significant step forward in diabetes management. HbA1c testing measures glycated hemoglobin, formed when glucose binds to the protein component of hemoglobin, the oxygen-transporting molecule present in red blood cells. Measuring the percentage of glycated hemoglobin in relation to total hemoglobin provides an index of the amount of glucose in the body. Normal HbA1c levels are between four percent and 5.6 percent, while measurements between 5.7 percent and 6.4 percent indicate prediabetes and rates above 6.5 percent signify diabetes.³ 

The use of HbA1c for diabetes management emerged in clinical laboratories around 1977. This was almost a decade after Samuel Rahbar, MD, PhD, discovered an “abnormal, fast-moving hemoglobin band” in a patient sample while looking for hemoglobin variants.⁴ He reviewed the patient’s history and found she was diabetic.⁵ This led Dr. Rahbar to screen an additional 47 patients, all of who had the same hemoglobin.⁵ The initial discovery served as the basis for subsequent work identifying HbA1c as a significant clinical biomarker for longer-term glycemic control and paved the way for the type of commercially developed assays still in use today. It was not until just over 40 years after Dr. Rahbar’s initial discovery, however, in 2010, that manufactured tests would become standardized to the point that the American Diabetes Association would recommend HbA1c testing as a standard of medical care in diabetes.⁶

HbA1c testing has been cited as one of the two most important advances in diabetes management. The other is portable blood glucose meters, used by people with diabetes to self-monitor their glucose levels.⁷ These portable glucose monitors are now giving way to a new generation of wearable products designed to provide patients with continuous glucose monitoring. While these technologies can keep diabetic patients and their physicians apprised of glucose levels, and thereby help minimize the sequelae of suboptimal blood glucose control, they do not replace HbA1c testing’s broader clinical utility; they support diabetes management, but they are not used to diagnose the disease. HbA1c, on the other hand, can be used for both management and initial diagnosis, thereby helping to identify previously undiagnosed patients and underscoring its relevance even in the face of increasing use of continuous glucose monitoring.  

HbA1c testing and blood glucose monitoring

Because the typical lifespan of a red blood cell is eight to 12 weeks, HbA1c testing provides a longer-term representation of overall blood glucose levels. Fasting plasma glucose and blood glucose testing, in contrast, offer information about a patient’s glucose levels at a given moment. The two types of tests complement each other, and both are used to monitor patients with either type 1 or type 2 diabetes. Type 1 diabetes occurs when a patient’s pancreas is unable to produce insulin. For those with type 2 diabetes, the pancreas can produce insulin, but either the body cannot effectively use it, or the amounts may be inadequate to support the body’s needs. Glucose and HbA1c measurements work together to provide both a short-term snapshot and longer-term picture of patient glucose levels.

Managing both long-term and short-term glucose levels is critical. Poor long-term control of blood glucose levels is associated with complications such as peripheral vascular disease, blindness, and kidney disease, whereas short-term low blood glucose causes symptoms that include lightheadedness, shakiness, and weakness.⁸ Untreated, hypoglycemia may result in shock and ultimately, death.

Three things laboratories need to know

While HbA1c testing has significantly advanced diabetes diagnosis, monitoring, and management, there are often under-recognized factors that can undermine testing accuracy. These include hemoglobin variants, red blood cell lifespan, and lipemia. Understanding these issues permits greater confidence in results for laboratorians and clinicians at the forefront of patient diabetes diagnosis and monitoring.  

Hemoglobin variants

The hemoglobin molecule is comprised of oxygen-carrying heme and four globin subunits. In normal adult hemoglobin, HbA, the globin subunits are two α and two β chains. Fetal hemoglobin (HbF), in contrast, is comprised of two α and two γ chains. Variant hemoglobins are those in which a germline DNA mutation results in a change in the amino acid sequence of a globin chain. These mutations may affect the glycation of hemoglobin, potentially impacting HbA1c results. The most common hemoglobin variants are HbS, HbE, HbC, and HbD, all of which involve the substitution of a single β-chain amino acid.⁹ HbF elevation may also be present in patients with variant hemoglobins.¹⁰ In some patients, the presence of a variant hemoglobin is clinically evident, so laboratorians and clinicians will likely recognize the limitations of HbA1c testing in those instances. Sickle cell anemia, for instance, affects one in 12 African Americans and one in 100 Hispanic Americans or Latinos,9 and presents with severe anemia and other symptoms.  

Hemoglobin E, on the other hand, is common in people from Southeast Asia,¹¹ and may not be clinically evident. A variant should be suspected if HbA1c results are inconsistent with results of blood glucose monitoring, as assays vary in their performance in patients with variant hemoglobins.¹⁰  

Red blood cell lifespan

In addition to variability in hemoglobin glycation, patients with variant hemoglobins may also experience shortened red blood lifespan and increased red blood cell turnover. As noted above, in some patients the presence of a condition that results in increased red blood cell turnover is clinically evident, so laboratorians and clinicians may recognize the limitations of HbA1c testing in those instances. In other instances, increased red blood cell turnover may not be clinically evident or suspected, or the clinician may not be aware of the potential for discrepancy between blood glucose and HbA1c levels. Note that hemolytic anemias may be either congenital (such as thalassemias) or acquired (thrombotic thrombocytopenia purpura or autoimmune hemolytic anemia). Thalassemias are a type of hemoglobinopathy that may occur together with a variant hemoglobin, further complicating the clinical picture.¹¹

In a world that is becoming increasingly migratory, it is helpful for laboratorians to stay mindful of the makeup of patient populations in their regions. Those patients with conditions or variants that affect red blood cell turnover or hemoglobin may require alternative tests. Information is available from the National Glycohemoglobin Standardization Program.¹²

Finally, any red blood cell transfusion for any reason may affect HbA1c results.¹³ Again, discrepancies between HbA1c and blood glucose monitoring should be investigated, and a blood transfusion history established.  

Lipemia

Lipemia refers to an overabundance of emulsified fat, or lipids, in the blood. Some individuals have high baseline lipid levels—typically hypertriglyceridemia—but postprandial lipemia is also seen in otherwise healthy patients following the consumption of a high-fat meal. Any assay that relies on a turbidimetric readout, including some HbA1c assays, may be affected by high levels of lipemia, as these assays rely on changes in light transmission through the sample. Importantly, although clinicians are generally aware of the need to run certain tests like fasting glucose and lipid profiles on fasting or, at least, non-postprandial samples, they are not necessarily aware of the potential for interference with turbidometric assays and are unlikely to know which assays are turbidometric.

Recent research suggests that enzymatic and immunoassay HbA1c assays can be vulnerable to lipemia interference.¹⁴ The choice of assay is driven by the workflow needs of the laboratory as well as the patient population. Immunoassays are popular due to their relatively low cost and ease of incorporation into the remainder of laboratory testing, but as assays vary in their susceptibility to interference from variant hemoglobins, laboratories may also consider the expected frequency of the various common variant hemoglobins in their community.¹²

Investigations of discrepancies between HbA1c and glucose monitoring should include an assessment of a particular assay’s susceptibility to lipemia interference, and, potentially, measurement of patient triglyceride levels. Postprandial lipemia may be avoided by advising patients to avoid a high-fat meal prior to phlebotomy.

Summary

HbA1c testing is an important tool in the fight against diabetes. As with all in vitro diagnostic tests, preanalytic and analytic issues can have an impact on accuracy. For HbA1c assays, issues to consider include hemoglobin variants, red blood cell turnover, and lipemia. Clinicians and laboratorians can work together to help ensure the accuracy of results, strengthening confidence in patient care decisions.

REFERENCES

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10. National Institutes of Health (NIH), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). “Sickle Cell Trait & Other Hemoglobinopathies and Diabetes (For Providers). June 2014. https://www.niddk.nih.gov/health-information/diagnostic-tests/sickle-cell-trait-hemoglobinopathies-diabetes. Accessed: 5 April 2019.
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12. National Glycohemoglobin Standardization Program (NGSP). “HbA1c Assay Interferences.” April 19, 2019. http://www.ngsp.org/interf.asp. Accessed: 6 June 2019.
13. Spencer DH, Grossman BJ and Scott MG. “Letter to the Editor: Red Cell Transfusion Decreases Hemoglobin A1c in Patients with Diabetes.” Clin Chem. 2011; 57:344–346.
14. Parker ML and Yip PM. “HbA1c Platforms are Variably Affected by Increasing Lipemia.” Abstract A-288. 70^th AACC Annual Scientific Meeting Abstracts, 2018.