Glycated hemoglobin

History

Hemoglobin A1c was first separated from other forms of hemoglobin by Huisman and Meyering in 1958 using a chromatographic column. It was first characterized as a glycoprotein by Bookchin and Gallop in 1968. Its increase in diabetes was first described in 1969 by Samuel Rahbar et al. The reactions leading to its formation were characterized by Bunn and his coworkers in 1975.

The use of hemoglobin A1c for monitoring the degree of control of glucose metabolism in diabetic patients was proposed in 1976 by Anthony Cerami, Ronald Koenig and coworkers.

Principle

Glycation of proteins is a frequent occurrence, but in the case of hemoglobin, a nonenzymatic reaction occurs between glucose and the N-end of the beta chain. This forms a Schiff base which is itself converted to 1-deoxyfructose. This rearrangement is an example of an Amadori rearrangement.

When blood glucose levels are high, glucose molecules attach to the hemoglobin in red blood cells. The longer hyperglycemia occurs in blood, the more glucose binds to hemoglobin in the red blood cells and the higher the glycated hemoglobin.

Once a hemoglobin molecule is glycated, it remains that way. A buildup of glycated hemoglobin within the red cell, therefore, reflects the average level of glucose to which the cell has been exposed during its life-cycle. Measuring glycated hemoglobin assesses the effectiveness of therapy by monitoring long-term serum glucose regulation.

A1c is a weighted average of blood glucose levels during the life of the red blood cells (120 days). Therefore, glucose levels on days nearer to the test contribute substantially more to the level of A1c than the levels in days further from the test.

This is also supported by data from clinical practice showing that HbA1c levels improved significantly after 20 days from start or intensification of glucose-lowering treatment.

Measuring HbA1c

A number of techniques are used to measure hemoglobin A1c.

Laboratories use:

High-performance liquid chromatography (HPLC): The HbA_1c result is calculated as a ratio to total hemoglobin by using a chromatogram.

Immunoassay

Enzymatic

Capillary electrophoresis

Boronate affinity chromatography

Point of care (e.g., doctor's office) devices use:

Immunoassay

Boronate affinity chromatography

In the United States, HbA_1c testing laboratories are certified by the National Glycohemoglobin Standardization Program (NGSP) to standardise them against the results of the 1993 Diabetes Control and Complications Trial (DCCT). An additional percentage scale, Mono S is in use by Sweden and KO500 is in Japan.

Switch to IFCC units

The American Diabetes Association (ADA), European Association for the Study of Diabetes (EASD) and International Diabetes Federation (IDF) have agreed that, in the future, HbA_1c is to be reported in the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) units. IFCC reporting was introduced in Europe except for the UK in 2003; the UK carried out dual reporting from 1 June 2009 until 1 October 2011.

Conversion between DCCT and IFCC is by the following equation:

I F F C H B A 1 c mmol mol = [ D C C T H B A 1 c ( % ) − 2.14 ] × 10.929

Interpretation of results

Laboratory results may differ depending on the analytical technique, the age of the subject, and biological variation among individuals. Two individuals with the same average blood sugar can have HbA_1c values that differ by as much as 3 percentage points.

Results can be unreliable in many circumstances, for example after blood loss, after surgery, blood transfusions, anemia, or high erythrocyte turnover; in the presence of chronic renal or liver disease; after administration of high-dose vitamin C; or erythropoetin treatment. In general, the reference range (that found in healthy young persons), is about 30–33 mmol/mol (4.9–5.2 DCCT %). The mean HbA_1c for diabetics type 1 in Sweden in 2014 was 63 mmol/mol (7.9 DCCT%) and for type 2, 61 mmol/mol (7.7 DCCT%).

Higher levels of HbA_1c are found in people with persistently elevated blood sugar, as in diabetes mellitus. While diabetic patient treatment goals vary, many include a target range of HbA_1c values. A diabetic person with good glucose control has a HbA_1c level that is close to or within the reference range.

The International Diabetes Federation and the American College of Endocrinology recommend HbA_1c values below 48 mmol/mol (6.5 DCCT %), while the American Diabetes Association recommends HbA_1c be below 53 mmol/mol (7.0 DCCT %) for most patients. Recent results from large trials suggest that a target below 53 mmol/mol (7 DCCT %) for older adults with type 2 diabetes may be excessive: Below 53 mmol/mol (7 DCCT %) the health benefits of reduced A1C become smaller, and the intensive glycemic control required to reach this level leads to an increased rate of dangerous hypoglycemic episodes.

A retrospective study of 47,970 old type 2 diabetes patients found that patients with an HbA_1c more than 48 mmol/mol (6.5 DCCT %) had an increased mortality rate, but a later international study contradicted these findings.

A review of the UKPDS, ACCORD (Action to Control Cardiovascular Risk in Diabetes), ADVANCE and VADT (Veterans Affairs Diabetes Trials) estimated that the risks of the main complications of diabetes (diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, and macrovascular disease) decreased by approximately 3% for every 1 mmol/mol decrease in HbA_1c.

However, a trial by ACCORD designed specifically to determine whether reducing HbA_1c below 6.0% using increased amounts of medication would reduce the rate of cardiovascular events found higher mortality with this intensive therapy, so much so that the trial was terminated 17 months early.

Practitioners must consider an individual patient's health, their risk of hypoglycemia, and their specific health risks when setting a target HbA_1c level. Because patients are responsible for averting or responding to their own hypoglycemic episodes, the patient's input and the doctor's assessment of the patient's self-care skills are also important.

Persistent elevations in blood sugar (and, therefore, HbA_1c) increase the risk of long-term vascular complications of diabetes such as coronary disease, heart attack, stroke, heart failure, kidney failure, blindness, erectile dysfunction, neuropathy (loss of sensation, especially in the feet), gangrene, and gastroparesis (slowed emptying of the stomach). Poor blood glucose control also increases the risk of short-term complications of surgery such as poor wound healing.

Lower-than-expected levels of HbA_1c can be seen in people with shortened red blood cell lifespan, such as with glucose-6-phosphate dehydrogenase deficiency, sickle-cell disease, or any other condition causing premature red blood cell death. Blood donation will result in rapid replacement of lost RBCs with newly formed red blood cells. Since these new RBCs will have only existed for a short period of time, their presence will lead HbA_1c to underestimate the actual average levels. There may also be distortions resulting from blood donation which occurred as long as two months before due to an abnormal synchronization of the age of the RBCs, resulting in an older than normal average blood cell life (resulting in an overestimate of actual average blood glucose levels). Conversely, higher-than-expected levels can be seen in people with a longer red blood cell lifespan, such as with Vitamin B₁₂ or folate deficiency.

The approximate mapping between HbA_1c values given in DCCT percentage (%) and eAG (estimated average glucose) measurements is given by the following equation:

eAG(mg/dl) = 28.7 × A1C − 46.7
eAG(mmol/l) = 1.59 × A1C − 2.59
Data in parentheses are 95% confidence intervals

The 2010 American Diabetes Association Standards of Medical Care in Diabetes added the HbA_1c ≥ 48 mmol/mol (≥6.5 DCCT %) as another criterion for the diagnosis of diabetes.

Indications and use

Glycated hemoglobin testing is recommended for both checking the blood sugar control in people who might be pre-diabetic and monitoring blood sugar control in patients with more elevated levels, termed diabetes mellitus. There is a significant proportion of people who are unaware of their elevated HbA_1c level before they have blood lab work. For a single blood sample, it provides far more revealing information on glycemic behavior than a fasting blood sugar value. However, fasting blood sugar tests are crucial in making treatment decisions. The American Diabetes Association guidelines are similar to others in advising that the glycated hemoglobin test be performed at least twice a year in patients with diabetes who are meeting treatment goals (and who have stable glycemic control) and quarterly in patients with diabetes whose therapy has changed or who are not meeting glycemic goals.

Glycated hemoglobin measurement is not appropriate where there has been a change in diet or treatment within 6 weeks. Likewise, the test assumes a normal red blood cell aging process and mix of hemoglobin subtypes (predominantly HbA in normal adults). Hence, people with recent blood loss, hemolytic anemia, or genetic differences in the hemoglobin molecule (hemoglobinopathy) such as sickle-cell disease and other conditions, as well as those that have donated blood recently, are not suitable for this test.

Due to glycated hemoglobin's variability (as shown in the table above), additional measures should be checked in patients at or near recommended goals. People with HbA_1c values at 64 mmol/mol or less should be provided additional testing to determine whether the HbA_1c values are due to averaging out high blood glucose (hyperglycemia) with low blood glucose (hypoglycemia) or the HbA_1c is more reflective of an elevated blood glucose that does not vary much throughout the day. Devices such as continuous blood glucose monitoring allow people with diabetes to determine their blood glucose levels on a continuous basis, testing every few minutes. Continuous use of blood glucose monitors is becoming more common, and the devices are covered by many health insurance plans but not by Medicare in the United States. The supplies tend to be expensive, since the sensors must be changed at least weekly. Another test that is useful in determining if HbA_1c values are due to wide variations of blood glucose throughout the day is 1,5 Anhydroglucitol, also known as GlycoMark. GlycoMark reflects only the times that the person experiences hyperglycemia above 180 mg/dL over a two-week period.

Concentrations of hemoglobin A1 (HbA1) are increased, both in diabetic patients and in patients with renal failure, when measured by ion-exchange chromatography. The thiobarbituric acid method (a chemical method specific for the detection of glycation) shows that patients with renal failure have values for glycated hemoglobin similar to those observed in normal subjects, suggesting that the high values in these patients are a result of binding of something other than glucose to hemoglobin.

In autoimmune hemolytic anemia, concentrations of hemoglobin A1 (HbA1) is undetectable. Administration of prednisolone (PSL) will allow the HbA1 to be detected. The alternative fructosamine test may be used in these circumstances and it also reflects an average of blood glucose levels over the preceding 2 to 3 weeks.

All the major institutions like International Expert Committee Report, drawn from the International Diabetes Federation (IDF), the European Association for the Study of diabetes (EASD), and the American Diabetes Association (ADA), suggests the HbA_1c level of 48 mmol/mol (6.5 DCCT %) as a diagnostic level. The Committee Report further states that, when HbA_1c testing cannot be done, the fasting and glucose tolerance tests be done.

Diagnosis of diabetes during pregnancy continues to require fasting and glucose tolerance measurements for gestational diabetes, and not the glycated hemoglobin.

Modification by exercise training

A meta-analysis of research done to identify the effect of two different kinds of training programs (combined aerobic and eccentric resistance exercise program and aerobic exercise only) on the glycated hemoglobin levels of individuals with type 2 diabetes mellitus (T2DM) found that the effect of combining resistance exercise with aerobic exercise improved the glucose control more than just the aerobics alone, but it should be noted that BMI of the resistance plus aerobic exercise group decreased more than the aerobic only group.

The average effect of the training programs included reductions of glycated hemoglobin of 9 mmol/mol (0.8 percentage points), which was a result similar to that of long-term diet and drug or insulin therapy (which result in a reduction of 6.5–9.0 mmol/mol (i.e. 0.6–0.8 points)).

Standardization and traceability

Hemoglobin A1c is now standardized and traceable to IFCC methods HPLC-CE and HPLC-MS.

A new unit (mmol/mol) is used as part of this standardization. The standardized test does not test for iodine levels in the blood. Hypothyroidism or iodine supplementation are known sources that artificially raise the A1c number.