In adults, hemoglobin A (Hb A) constitutes about 97%-98% of normal hemoglobin; the remainder includes about 2.5% hemoglobin A2 and about 0.5% hemoglobin F (Hb F). About 6%-7% of Hb A consists of Hb A molecules that have been partially modified by attachment of a glucose molecule to the terminal valine amino acid of the globin beta chain. This process is called “glycosylation,” and this particular glycosylated hemoglobin is called “hemoglobin A1” (Hb A1). Although Hb A1 comprises the great majority of glycosylated hemoglobin under usual conditions, glycosylation to some degree may occur at other locations in the globin chain and in other hemoglobins besides Hb A. The sum of the various glycosylation activities occurring in all hemoglobins (normal or abnormal) in the patient is known as total glycosylated hemoglobin.

Glycosylation of hemoglobin occurs during exposure of red blood cells (RBCs) to plasma glucose; hemoglobin and glucose can form a bond that initially is labile but then becomes stable. Once stable bonding occurs, it is very slowly and poorly reversible. In Hb A1, the labile bonding fraction normally constitutes about 10% of total glucose bonding. Formation of Hb A1 occurs very slowly during the entire 120-day life span of the RBC, and the number of Hb A molecules affected by glycosylation depends on the degree and duration of RBC exposure to glucose. Hemoglobin A1 is actually composed of three hemoglobins: A1a, A1b, and A1c. Of these, Hb A1c is about 70% glycosylated, whereas the other two are less than 20% glycosylated. In addition, Hb A1c constitutes about 60%-70% of total Hb A1. Since Hb A1 comprises the majority of the predominant glycosylated Hb A fraction, under usual conditions Hb A1c therefore represents the majority of glycosylated hemoglobin. Because of this relationship the term glycosylated hemoglobin (or glycoHb) has been used for both Hb A1 and its major component Hb A1c, which sometimes is confusing. The components of total glycosylated Hb, Hb A1, and Hb A1c are shown in the box. There is a strong correlation between all three parameters, and, in most circumstances, any of the three can provide clinically useful information. However, there are differences, and in some cases one or the other is more advantageous.

Components of Hb A1, A1c, and Total GlycoHb

Hb A1c
Glycosylated Hb A1c
Nonglycosylated Hb A1c
Hb A1
Glycosylated Hb A1c
Nonglycosylated Hb A1c
Hb A1a + Hb A1b
Negatively charged non-A glycosylated hemoglobins*
Total Glycosylated Hb (Affinity method)
Glycosylated Hb A1c
Nonglycosylated Hb A1c
Hb A1a + Hb A1b
Negatively charged non-A glycosylated hemoglobins*
Positively charged non-A glycosylated hemoglobins†
Hb A glycosylated elsewhere than Hb A1 sites
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*Hb Bart’s, F, G, H, I, J (Baltimore), M, and N.
†Hb A2, C, D, E, and S.

An increase in glycoHb quantity can be produced by recent very high short-term increases in blood glucose (in which case labile bonding is primarily affected), but is most often caused either by relatively continual elevation of blood glucose or by intermittent elevations that are frequent enough to produce abnormally high average glucose levels (in both of these cases stable glycosylation is primarily affected). A measurable increase in glycosylated (stable) hemoglobin begins about 2-3 weeks (literature range, 1-4 weeks) after a sustained increase in the average blood glucose level and takes at least 4 weeks to begin decreasing after a sustained decrease in the average blood glucose level. GlycoHb assay represents the averaged blood glucose levels during the preceding 2-3 months (literature range, 1-4 months). In contrast, blood glucose increases or decreases of “moderate” (100 mg/100 ml; 5.55 mmol/L) degree that occur within the 3 days just before Hb A1 measurement add sufficient labile component so as to constitute as much as 15% (range, 12%-19%) of the glycoHb result. Spontaneous sudden decreases in blood glucose of this magnitude are not common, so that under most circumstances a normal glycoHb level is good evidence of relatively normal average blood glucose during at least the preceding 4 weeks. Most of the clinical problems with labile bonding component occur when it produces false increases in glycoHb levels. In summary, an elevated glycoHb level is most often due to long-term average blood glucose elevation over the preceding 2-3 months, but the possibility exists for elevation due to marked short-term blood glucose increase if an assay method is used that is not specific for stable bonding.

GlycoHb measurement has been used to monitor effectiveness of (long-term) diabetic therapy, to monitor patient compliance with therapy, and to differentiate between short-term stress-related glucose tolerance abnormality (e.g., acute myocardial infarction) and diabetes. Of these, the most widely accepted indications are monitoring of diabetic therapy effectiveness and monitoring of patient compliance. GlycoHb assay has also been used to diagnose diabetes mellitus, but this is controversial.

Laboratory methods

As noted previously, glycoHb can be measured as either total glycoHb, Hb A1, or Hb A1c (since most of total glycoHb is Hb A1 and most of Hb A1 is Hb A1c). The majority of commercially available kits measure Hb A1 and report the result as a percentage of total hemoglobin. There are a variety of assay methods. Currently, most commercial kits assaying Hb A1 or A1c use some method involving ion exchange resin. Less than 20% use agar electrophoresis or high performance liquid chromatography. Total glycoHb is assayed by a special boronic acid resin that reacts only with the stable glycated fraction and does not need pretreatment. There are surprisingly few evaluations of different glycoHb kits. In some, it is difficult to tell what they are measuring. In several kits evaluated in my laboratory there was significant variation in reproducibility and accuracy.

Sources of error. Most ion-exchange resin-based kits do not differentiate between labile and stable glucose bonding to hemoglobin. Certain techniques available can eliminate the labile fraction before testing the patient serum. Many hemoglobins can form glycoHb to some extent. However, with some ion-exchange resin methods for A1 or A1c, positively charged non-A hemoglobins do not elute from the resin with Hb A1 or A1c but instead remain on the resin with nonglycosylated Hb A (see the box). These hemoglobins (such as Hb S and HbC) may produce glycoHb assay values that are less than true levels because these abnormal hemoglobins have a glycosylated component that is not being measured along with Hb A1. On the other hand, negatively charged non-A hemoglobins such as Hb F and Hb H elute from the resin in the same fraction as Hb A1. Therefore, an increased Hb F value or the presence of Hb H could falsely increase Hb A1 or A1c values since they are included in Hb A1 assay. The hemoglobin F value may be increased in young infants, in up to 17% of pregnant women, and in patients with some of the hemoglobinopathies. Therefore, it may be advantageous to use a method such as total glycoHb by boronic affinity when there are significant numbers of patients who are not of northern European descent. Some of the resin methods are affected by temperature changes, and in some, chronic renal failure has been reported to produce falsely high results. A few reports have described false increase with aspirin, alcoholism, and lead poisoning. It is necessary to find out what will falsely increase or decrease any A1 or A1c method. Total glycoHb measure by boronic acid chromatography includes the results of abnormal hemoglobin glycation as well as Hb A glycation and is not affected by renal failure, aspirin, or temperature fluctuations. Hemolytic anemia may produce falsely low glycoHb values with any method because hemolysis results in a shortened RBC life span and RBCs therefore are not exposed to blood glucose as long as a normal RBC. This is accentuated by bone marrow reticulocyte response since the reticulocytes are young cells with no glucose exposure. Frequent episodes of hypoglycemia might decrease glycoHb levels somewhat. Finally, there is some difficulty in calibration of assay kits because primary standards (i.e., material with substance values that are known with absolute certainty) are not available.

In summary, glycoHb assay provides information of great value in the treatment of diabetes and in certain cases may help in the diagnosis. However, the sensitivity and reliability of some commercial kits still need improvement.