Hemoglobin A1c and Glycated Hemoglobins

Hemoglobin A1c and Glycated Hemoglobins
By William H. Wong, Ph.D., December 7, 1999

Diagnostic Laboratory Services has been performing glycated hemoglobin assays since 1990. We currently perform two glycohemoglobin assays: one reporting total glyco-hemoglobin (and a calculated hemoglobin A1c) using affinity chromatography, and one reporting measured hemoglobin A1c using ion-exchange HPLC. Both methods are certified by the National Glyco-hemoglobin Standardization Program (NGSP) as traceable to and standardized against the Diabetes Control and Complications Trial (DCCT) reference method.

In order to reduce the confusion as to which assay is preferred and which result is to be used, we have standardized the methodology to use only the DCCT reference method – Hemoglobin A1c by ion-exchange HPLC. We have completed the evaluation and validation of a fully automated ion-exchange HPLC assay (BioRad Variant II) for the determination of hemoglobin A1c. Because it is now fully automated, the reproducibility of the test is also substantially improved.

Effective December 7, 1999, Glycohemoglobin (Test Code 418) will be discontinued and will automatically be converted to Hemoglobin A1c by HPLC (Test Code 4779). For diabetic monitoring purposes, please refer to the recommended reference guidelines for Total GHb and HbA1c on this page.

If you have any questions, please contact one of us. Thank you for your continued support.

Terminology

Glycohemoglobin (GHb, glycated hemoglobin, glycosylated hemoglobin) is a generic term for hemoglobin bound irreversibly (ketoamine form) to glucose. Often, the term is used to mean total glycated hemoglobin, and sometimes to mean hemoglobin A1c.

Total glycated hemoglobin (Total GHb) refers to all the glycated hemoglobins, including glycated hemoglobin variants. Total glycated hemoglobin is usually determined by affinity chromatography or immunassays.

Hemoglobin A1c (HbA1c) is the major subfraction of the glycated normal hemoglobin (HbA1). Determination of HbA1c is usually achieved by ion-exchange HPLC or gel electrophoresis.

Glycated Hemoglobins

Glycated hemoglobins are hemoglobin components formed through a two-step non-enzymatic reaction between hemoglobin and blood glucose. The first step consists of the formation of a reversible aldimine form of hemoglobin to glucose linkage. In the second step, the labile aldimine form is converted slowly to the stable and irreversible ketoamine form through an Amadori rearrangement.

The level of glycated hemoglobins in the blood is directly related to the average blood glucose levels over the life span of the hemoglobin in the circulation. Since the half-life of red blood cells is about 120 days, a single determination of glycated hemoglobin reflects the average blood glucose level during the preceding 8 to 12 weeks. The test is therefore a very good monitor for long-term (2 to 3 months) blood glucose control in patients with diabetes mellitus.

Reference Guidelines

In patients with uncontrolled diabetes, the % of glycated hemoglobin is substantially higher than in diabetics in good control and in non-diabetics. The determination of a glycated hemoglobin level may therefore also assist in the initial diagnosis of diabetes, or it may be used to indicate the degree of long-term diabetic control in diabetic patients. The significance of a low glycated hemoglobin level has not been established.

Correlation with Mean Blood Glucose Levels

A single fasting blood glucose measurement only gives an indication of the patient’s immediate past (last 1 to 2 hours) condition, and may not represent the true status of blood glucose regulation. In contrast, the level of glycated hemoglobin is directly related to the average glucose concentration over the lifespan of the hemoglobin in the circulation.

Various formulae have been proposed to demonstrate the correlation between mean blood glucose (MBG) and Hemoglobin A1c (HbA1c). The following is one from Nathan, et al, N Engl J Med (1984).

MBG = 33.3 * HbA1c – 86

To verify the correlation, the mean blood glucose level for each patient was obtained as the average of up to 4 daily determinations over a period of 2 months (the average of over 200 glucose readings). Hemoglobin A1c was determined by ion-exchange HPLC at the end of the two-month period. Note that the mean blood glucose value is the average glucose level over the past 60 days, and not the glucose value of the specimen obtained at the same time as the HbA1c.

Methodology Options

There are currently four main techniques for determining glycated hemoglobins:

1. Cation-exchange chromatography – separates hemoglobins using HPLC based on net charge as a result of glycation;
2. Gel electrophoresis;
3. Affinity chromatography – separates total glycated hemoglobins by binding to solid-phase dihydroxyborate;
4. Immunoassay – based on binding to specific antibodies.

DLS Methodology

DLS will be using the Bio Rad Laboratories’ Variant II program. This method utilizes the ion-exchange high-performance liquid chromatography (HPLC) for the separation of hemoglobin A1a, A1b, and A1c. The program is certified by the National Glycohemoglobin Standardization Program (NGSP) as traceable to the Diabetes Control and Compliance Trial (DCCT) reference method. The method is fully automated, thus resulting in excellent precision, with a typical inter-assay variation less than 4%.

However, the method does not automatically include any glycated hemoglobin variants (Hb F, S, C, E and others). In the presence of any variant hemoglobin, HbA1c levels may need to be corrected by including the glycated fraction of that variant hemoglobin.

By using a method that is NGSP certified, any of the conclusions and recommendations from the DCCT can be directly applied to the patient’s hemoglobin A1c results. The DCCT clearly demonstrated that, compared with conventional therapy, a regimen of intensive therapy to maintain near-normal blood glucose and hemoglobin A1c levels significantly reduces the risks of the development or progression of microvascular and neuropathic complications of IDDM.

References

1. Diabetes in the 1980’s: Challenges for the Future. Report of the National Diabetes Advisory Board.
   U.S. Department of Health and Human Services, NIH Pub. No. 82-2143, Washington D.C., 1982.
2. Weykamp, CW et al. Clin Chem 39:1717-1723 (1993).
3. American Diabetes Association, Diabetes Care 20 Sup.1:S5-S13 (1997).
4. Klenk DC et al. Clin Chem 28:2088-2094 (1982).
5. Nathan, et al. N Engl J Med 310:341-346 (1984).

Ordering Information

Testing Site: DLS Special Chemistry.
Methodology: Cation-exchange chromatography on HPLC

Specimen Requirements:

• EDTA Whole Blood (Lavender top tube), 2.0 mL (minimum 1.0 mL)

Storage and Specimen Stability:

• Stable at room temperature for up to 12 hours.
• Refrigerated below 4°C for up to 7 days.
• Frozen (below -20°C) for long-term storage.

Reference Ranges

Procedure Codes: HbA1c (HPLC) 83036.