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Hemoglobin A1c and the Estimated Average Glucose

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Published: July 2009

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Dr. Saenger provides an overview of the diagnosis of diabetes and monitoring of the diabetic patient and discuss the advantages and disadvantages of hemoglobin A1c and fasting plasma glucose testing. She defines newly released recommendations from the American Diabetes Association and discusses the use and reporting of the estimated average glucose.

Presenter: Amy K. Saenger, PhD

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Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice.

Our presenter for this program is Amy K. Saenger, PhD, Director of the Central Clinical Laboratory in the Department of Laboratory Medicine & Pathology at Mayo Clinic in Rochester, Minnesota. Dr. Saenger will provide an overview of the diagnosis of diabetes and monitoring of the diabetic patient and discuss the advantages and disadvantages of hemoglobin A1c and fasting plasma glucose testing. She will define newly released recommendations from the American Diabetes Association and discuss the use and reporting of the estimated average glucose.

I will be discussing hemoglobin A1c testing and reporting of the estimated average glucose. This is a subject that has received much attention in both the clinical and laboratory realm due to the fact that there are major implications for measuring an accurate and precise HbA1c.

Diagnosis of Diabetes

The current diagnosis of diabetes includes one of the following: a fasting plasma glucose greater than or equal to 126 mg/dL, symptoms of hyperglycemia and a random plasma glucose greater than or equal to 200 mg/dL or a 2 hour glucose concentration great than or equal to 200 mg/dL after the oral glucose tolerance test. It is also recommended that confirmatory testing be performed on a different day unless there is clear evidence of hyperglycemia.

Categories of Glucose Values

Glucose concentrations are further broken down into different categories, with a normal fasting plasma glucose defined as ≤100 mg/dL, impaired fasting glucose as 101-125 mg/dL, and a provisional diagnosis of diabetes can be made when the glucose is >=126 mg/dL. Corresponding categories of glucose using the oral glucose tolerance test are also noted on this slide.

Importance of Diagnosis

Previous research trial groups, including the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS) both demonstrated a strong relationship between the level of plasma glucose control for both type 1 and type 2 diabetes and the risk of retinal, renal, and neurological complications. Therefore, the critical importance on diagnosis of diabetes relates to the high prevalence of long term medical complications in diabetics, which in turn can have devastating effects on both the patients and place a burden on the health care system. It is estimated that one quarter of diabetics manifest complications at the time of diagnosis and there is an approximate 7 year gap between the onset of diabetes and clinical diagnosis.

Role of the Laboratory

The role of the laboratory in terms of chronic diabetes management includes measurement of glucose, glycated proteins such as hemoglobin A1c and fructosamine, and urinary proteins to evaluate for microalbuminuria.

Fasting Plasma Glucose

Measurement of the fasting plasma glucose has several advantages; glucose is easy to measure in terms of technical intervention and it is widely available on automated instruments. Only one specimen is needed, as opposed to timed specimens with the oral glucose tolerance test. In addition there is a single cutoff used for diagnosis, 126 mg/dL. It should also be mentioned that point of care analyzers are not sufficiently precise enough to be used for diagnosis of diabetes and should not be used for this purpose.

Disadvantages of using the fasting plasma glucose include that the patient must fast at least 8 hours, and preferably 12 hours; there is a diurnal variation in glucose, such that specimens should be drawn in the morning when values are at their peak and individuals will not be under diagnosed. These two issues both present an inconvenience for the patient. In addition, there is a large biological variability with a fasting glucose, with intraindividual CVs of 5-8% and interindividual CVs of 7-13%. Fasting plasma glucose is also considered to be less sensitive than the oral glucose tolerance test. There are preanalytical problems as well, and it is an often overlooked fact that use of a sodium fluoride collection tube does not prevent glucose degredation for the first 30-90 minutes after the specimen is drawn.

Hemoglobin A1c (HbA1c)

Hemoglobin A1c is another important analyte for monitoring the progress of a diabetic over time. Adult hemoglobin is comprised mainly of hemoglobin A, and to a lesser extent hemoglobin A2 and fetal hemoglobin in hematologically normal individuals. Hemoglobin A1c should not be confused with hemoglobin A1, as hemoglobin A1 consists of hemoglobin A1a, A1b and A1c; however, hemoglobin A1c makes up approximately 80% of hemoglobin A1. Hemoglobin A1c is also referred to as glycated hemoglobin, glycohemoglobins, and fast hemoglobin, due to its migration pattern on electrophoresis.

Glycation

Hemoglobin glycation occurs when glucose attaches to one or both of the N-terminal valines of the b-chain to form a Schiff’s base. The process is fast and unstable and proceeds to form an irreversible ketoamine through Amadori rearrangement. After a period of weeks, advanced glycation endproducts are formed and broken down.

HbA1c Concentration

Glycation and formation of the stable ketoamine is an irreversible process. The concentration of hemoglobin A1c depends upon several factors. The life span of the red blood cell and how long the hemoglobin A1c is exposed to glucose is a major determining factor. It is also thought that the permeability of the red blood cell to glucose influences the amount of glycation and could explain the discordance noted in some hematologically normal people with diabetes in whom A1c appears discordant from other measures of glycemic control. In general hemoglobin A1c concentrations represent glucose levels over 8-12 weeks. This is precluded in patients who have underlying anemias, hemolysis, B12 or folate deficiencies or hemoglobinopathies which may affect the lifespan of the red blood cells.

Diabetes Treatment Goals

There are many international diabetes organizations which have treatment goals based on the hemoglobin A1c concentration. In the United States, the most widely accepted goal is the American Diabetes Association recommendation of <7%, although this is adjusted based on the individual patients, as some patients need stricter goals and others need less stringent targets.

HbA1c Methods

Hemoglobin A1c methods can essentially be divided into two categories: those that measure HbA1c based upon charge and those that quantitate HbA1c by structure. A common charge based method utilizes cation-exchange high pressure liquid chromatography, where hemoglobin species elute from the cation-exchange column at different times, depending on their charge, with the application of buffers of increasing ionic strength. Changes or mutations in the hemoglobin molecule that result in any charge differences may or may not alter the normal elution time of those hemoglobin species when using cation-exchange chromatography; in general, charge based methods are considered to be more susceptible to interference from hemoglobin variants. Structural methods include boronate affinity chromatography, which separates glycated from non-glycated compounds and is least affected by the presence of variants. Immunoassays are another structural method commonly used in clinical laboratories and employ the use of antibodies which target the b-N-terminal glycated amino acid in the first 4-10 amino acids, depending upon the manufacturer. Interferences with immunoassays are rare. The biggest downfall in using a structural method for measuring A1c is the inability to detect the presence of hemoglobin variants and use of this method should be used with caution in populations where the prevalence of hemoglobin variants is high.

Hemoglobin Variants

In the United States, hemoglobin S is the most common variant, followed by hemoglobin C, then hemoglobin E, and hemoglobin D (Punjab/Los Angeles). Worldwide, hemoglobin variants follow this similar trend except hemoglobin E is more common than hemoglobin C. Many, but not all, HbA1c methods are unaffected by the presence of some or all of these variants. For patients who have no hemoglobin A, such as homozygous S or C patients, hemoglobin A1c measurement will be inaccurate and it is recommended that an alternate form of glycemia be used such as fructosamine. In these patients a shortened red blood cell lifespan is typically encountered, which leads to a falsely low hemoglobin A1c.

Hemoglobinopathies

Although there have been over 950 different variants identified, most have a benign phenotype and patients are not affected clinically. However, these variants can cause falsely increased or decreased hemoglobin A1c concentrations and can lead to difficulty in chromatographic interpretation. Boronate affinity methods will give an accurate hemoglobin A1c in these patients, as long as the hemoglobinopathy does not cause issues with red blood cell turnover.

Reporting HbA1c as an eAG

There has been much discussion lately in regards to reporting hemoglobin A1c as an estimated average glucose. After the DCCT and UKPDS trials it was clear there was a direct relationship between hemoglobin A1c and the mean blood glucose. The major advantage to reporting HbA1c as an eAG is that both physicians and patients understand glucose. Patients are familiar with dealing with glucose values from their home glucose meters. Until recently, there were not any reliable regression equations available to calculate an eAG.

HbA1c and Average Glucose

Last year there were two major papers published which involved clinical and analytical validation of the relationship between HbA1c and eAG.

Derivation of Estimated Average Glucose (eAG)

In this A1c Derived Average Glucose (ADAG) study, 507 diabetic and non-diabetic subjects were evaluated, although the majority of the patients were type 1 diabetics. A linear regression was calculated which directly used HbA1c to calculate the estimated average glucose. The correlation was 0.84 and was deemed significant.

HbA1c/eAG Table

The following table shows the relationship of the measured hemoglobin A1c with the calculated estimated average glucose.

US Diabetes Prevalence

Diabetes is currently the seventh leading cause of death in the developed world. The American Diabetes Association defines diabetes as a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. In the United States, it is estimated from recent statistics that 23.6 million people have diabetes. Approximately 18 million of those individuals are diagnosed with diabetes, with type 1 diabetes accounting for 5-10% of cases and type 2 diabetes accounting for the majority of newly diagnosed patients. However, it is recognized that there are still 5-6 million individuals who remain undiagnosed and potentially at risk for premature development of micro and macrovascular complications.

Limitations

There are some limitations noted with the A1c Derived Average Glucose study. There were a small number of ethnic groups included, and most of the subjects were Caucasian. There were no data in children, pregnant women, or patients with renal impairment. In addition, there was enough scatter around the hemoglobin A1c values that brings the concept of the glycation gap into the picture. It is hypothesized that some patients are high glycators and some are low glycators. This means although they have the same average blood glucose, the high glycator will have a much higher A1c than the low glycator. This is notable if you examine the confidence intervals around a hemoglobin A1c of 7%, which translates to an eAG of 154 mg/dL. The confidence intervals around the HbA1c is 6.7-9.2% and the eAG is anywhere from 123 to 185 mg/dL. This wide margin of error has potential clinical and analytical implications for interpretation.

Endorsement of eAG

Despite some of these shortcomings, reporting of the eAG has been endorsed by several clinical groups such as the American Diabetes Association and International Diabetes Foundation and laboratory medicine groups like the American Association of Clinical Chemistry and the International Federation of Clinical Chemistry.

HbA1c for Diagnosis of Diabetes

In addition to reporting the eAG, hemoglobin A1c has also been targeted for a potential role in the diagnosis of diabetes. The advantages to using HbA1c include that it gives a measurement of chronic hyperglycemia, based on the individuals red blood cell (RBC) lifespan. The standardization efforts from the National Glycohemoglobin Standardization Program have been largely successful and the accuracy of HbA1c is closely monitored by manufacturers and laboratories. No fasting is necessary to measure HbA1c and there is very low intraindividual variability with a CV of <2%. In addition it would be a single test which could be used for both diagnosis and monitoring of diabetes.

Disadvantages

Some arguments against using HbA1c for diagnosis of diabetes include the limited number of studies which have been performed thus far to derive an adequate cutoff. There are other conditions which alter HbA1c values including the presence of variants, uremia, transfusions, and anemias. Some would also argue there are analytical issues which remain unresolved. HbA1c is also a more expensive analyte to measure in the laboratory than glucose.

International Expert Committee Report on the Role of the A1c Assay in the Diagnosis of Diabetes

Very recently HbA1c was endorsed by an international expert committee to be used in the diagnosis of diabetes. A cutpoint of 6.5% was recommended based on the sensitivity and specificity of several studies. An elevated HbA1c should be confirmed with a repeat measurement except in those individuals who are symptomatic and also have an increased plasma glucose over 200 mg/dL. The terms pre-diabetes, impaired fasting glucose, and impaired glucose tolerance will be phased out to eliminate confusion. Patients who have an HbA1c >=6.0 but <=6.5% are still considered at risk for developing diabetes in the future. There is no endorsement of HbA1c for diabetes screening at this time.

HbA1c at Mayo Clinic

On April 28th, 2009 the Central Clinical Laboratory made a change to hemoglobin A1c testing. The previous methodology utilized an immunoassay performed on the Roche Modular system but a change was made due to issues with the manufacturer’s calibration of the assay. HbA1c testing is now performed on the BioRad Variant Turbo, a cation ion-exchange HPLC method. Reflex testing is performed using boronate affinity methodology if a result is unable to be obtained on the Variant Turbo. The HbA1c reference range also changed and is now 4-6% which is a decision based reference range from the American Diabetes Association, as opposed to the previous reference range which was determined by normal population sampling. Reporting of the eAG at the Mayo Clinic is still being debated at this time, however the conversion equation is reported in Mayo Access.

Conclusions

In conclusion, the optimal diagnostic criteria for diabetes are still being debated. Hemoglobin A1c has recently been endorsed by clinical groups for diagnosis of diabetes using a cutpoint of 6.5%. Reporting of the eAG at this time is still controversial and will vary from laboratory to laboratory depending on the preference of the physicians, pathologists, and endocrinologists.


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