Postscript

Introduction:

• Hemoglobin A1c (HbA1c; also called glycated hemoglobin) is one of several minor adducts of hemoglobin A.
• HbA1c arises from a posttranslational substrate-concentration-dependent reaction:
  • HbA1c is formed from hemoglobin A by the addition of a molecule of glucose to the N-terminal valine of one or both of the beta chains. This reaction is spontaneous, nonenzymatic, and irreversible.
  • HbA1c is formed at a rate dependent on the glucose concentration to which the red blood cell (RBC) is exposed.
• For decades, HbA1c has remained the standard biomarker for glycemic control, providing a measure of glycemic control over the previous 3 month period (the higher the glucose concentrations over the previous 2-3 months, the higher HbA1c will be)
  • Used in diagnosing patients with diabetes and prediabetes.
  • Used in guiding therapy in patients with diabetes and prediabetes.
• HbA1c is a measure of the percentage of hemoglobin molecules attached with glucose.
• As with all laboratory tests, HbA1c measurements are associated with variability.

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Definitions:

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What is hemoglobin A1c?

HbA1c is formed by the nonenzymatic condensation of glucose and the N-terminal valine residue of the beta-chain.

• Hemoglobin in adults includes:
  • Hemoglobin A (α2β2) – 97%
    • 90% no posttranslational modification
    • 5-7% with posttranslational modification
  • Hemoglobin A2 – 2.5% of the total
  • Fetal Hb – 0.5% of the total
• Normal adult hemoglobin molecule (HbA):
  • Consists of two α and two β chains (α2β2).
  • Makes up about 97% of most normal human adult hemoglobin.
• Other minor hemoglobin components may be formed by posttranslational modification of HbA:
  • Including negatively charged minor components, collectively designated HbA1:
    • Hemoglobin A1a – 0.5% of the total hemoglobin
    • Hemoglobin A1b – 0.5% of the total hemoglobin
    • Hemoglobin A1c – comprises 4-6% of the total hemoglobin (the most abundant minor hemoglobin component)
  • HbA1 also referred to as:
    • The “fast hemoglobins”
      • ‘‘fast’’ is derived from the fact that these components elute faster from a cation-exchange column than the other components.
      • The fractions were described in the order in which they were eluted from the column: HbA1a, HbA1b and HbA1c, respectively.
    • Glycated hemoglobins
• HbA1c is formed by the chemical condensation of hemoglobin and glucose (non-enzymatic reaction):
  • This process occurs slowly and continuously over the life span of erythrocytes, which is 120 days on average.
  • The rate of A1c formation is directly proportional to the average concentration of glucose within the erythrocyte during its lifespan – thus, HbA1c content of aged erythrocytes is significantly higher than that from young erythrocytes.
  • HbA1c represents a weighted mean of glucose levels during the preceding 3 month time period, with the glucose level of the preceding 30 days contributing more to the test result than glucose levels 90 to 120 days earlier. 
  • HbA1c has no known physiologic function.

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Clinical use:

• HbA1c measures the glycemic effect on hemoglobin over the preceding 3 months.
• HbA1c can be converted into an estimated average glucose: as a general rule, every 1% change in A1c is associated with an approximate 30 mg/dL change in estimated average glucose.
• According to the American Diabetes Association (ADA), diabetes is diagnosed at an A1C of greater than or equal to 6.5%:

• American Diabetes Association (ADA) recommended HbA1c target goals for nonpregnant adults with diabetes:
  • HbA1c < 7% (53 mmol/mol) is a reasonable goal for many nonpregnant adults without significant hypoglycemia. 
  • More stringent target, such as HbA1c < 6.5% (48 mmol/mol), may be reasonable if it can be achieved without significant hypoglycemia or other adverse effects of treatment (such as polypharmacy).
• Alternative markers, such as fructosamine, glycated albumin, and 1,5-anhydroglucitol may be considered when HbA1c values and glucose testing results are inconsistent and can be useful for reflecting shorter term changes in glycemia.

Guideline	Recommended target range
ADA	7% for the general population
SIGN	7% for the general population
AACE/ACE	6.5%
NICE	6.5% or 7%, depending on the patient’s treatment regimen
ICSI	<7% to less than 8% based on patient factors
VA/DoD	6% to 7% for patients with a life expectancy greater than 10 to 15 years
ACP	Clinicians should aim to achieve an HbA1c level between 7% and 8% in most patients with type 2 diabetes.

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Historical Background:

Date	Finding
Kunkel HG and Wallenuis G. Science. 1955;122:288.	Human adult hemoglobin could be separated into fast-, intermediate-, and slow-migrating components.
Allen DW et al. J Am Chem Soc. 1958;80:1628.	Named these chromatically fast-moving minor components HbA1a, HbA1b, and HbA1c. Hemoglobin A1C first recognized as a distinct minor hemoglobin of the human red blood cell.
Huisman TH and Dozy AM. J Lab Clin Med. 1962;60:302-319.	The first to report a 2 to 3 fold increase in the minor hemoglobin component in four diabetics.
Holmquist WR et al. Biochemistry. 1966;5:2489-2503.	Found that Hb A1c is structurally identical to Hb A except that an unidentified group is linked to the terminal amino group of the beta-chain by means of a Schiff base.
Bookchin RM et al. Biochem Biophys Res Comm. 1968;32: 86-93.	Showed that the N -terminal compound of the beta chain possessed an added hexose.
Rahba S. Clin Chim Acta. 1968;22:296-298.	Elevation in a minor hemoglobin in two diabetics, later confirmed this in 47 others. The minor hemoglobin component was subsequently identified as HbA1c. This discovery of a “diabetic hemoglobin” increased interest in gHb and promoted the development of clinically useful assay methods.
Trivelli LA et al. N Engl J Med. 1971;284:353-357.	used a simplified cation-exchange column method to measure gHb levels in a group of diabetic patients The cation exchanger used was Bio-Rex 70.
Bunn HF et al, Biochem Biophys Res Comm. 1975;67:103-109.	Identified the hexose as glucose and mannose in a ratio of 3 :1.
Koenig RJ and Cerami A. Proc. Nat. Acad. Sci. USA 1975;72:3687-3691.	Found that the concentration of HbA1c appeared to reflect the mean blood sugar level over the previous weeks.
Bunn FH et al. J Clin Invest. 1976; 57:1529-1659.	Hb A1c is slowly formed during the 120-day life-span of the erythrocyte, probably by a nonenzymatic process.
1977	The first commercial HbA1c method became available.
1985	WHO mentioned .the potential utility of HbA1c in diabetes care.
1988	The American Diabetes Association (ADA) first recommended using A1c
1990s	US Food and Drug Administration (FDA) began to approve drugs for the treatment of diabetes based on hemoglobin A1c (HbA1c) levels as the outcome
The Diabetes Control and Complications Trial (DCCT) published in N Engl J Med 1993;329:977– 86.	Showed that intensive therapy effectively delays the onset and slows the progression of diabetic retinopathy, nephropathy, and neuropathy in patients with IDDM. Demonstrated importance of HbA1c as a predictor of diabetes-related outcomes.
1994	The ADA started recommending specific A1c targets
The United Kingdom Prospective Diabetes Study (UKPDS) published in Lancet. 1998;352:837-53.	Showed that intensive blood-glucose control substantially decreased the risk of microvascular complications, but not macrovascular disease, in patients with type 2 diabetes.
International Expert Committee report on the role of the A1C assay in the diagnosis of diabetes. Diabetes Care 2009;32:1327–34.	2009 an international expert committee concluded that the available evidence supported the use of HbA1c for the diagnosis of diabetes.
American Diabetes Association. Standards of medical care in diabetes. Diabetes Care. 2010;33 Suppl 1:S11– 4.	The role of the A1c broadened in 2010 when the ADA added A1c ≥6.5 % as a diagnostic criterion for diabetes.

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Hemoglobin A1c measurements:

Also called the hemoglobin A1C, HbA1c, or glycohemoglobin test, the A1C test measures the amount of glycated hemoglobin, which is based on the attachment of glucose to hemoglobin over the typical 120-day life span of a red blood cell.

Several methods are available to measure glycosylated hemoglobin. Each method has specific advantages and disadvantages. Assays are based on either of two principles:

• Charge differences:
  • Methods based on charge differences depend on the extra negative charge that occurs when glucose is attached to the N-terminal valine of the HbA beta chain. At a slightly acidic pH , the minor hemoglobins are more negatively charged than HbA.
  • Subject to interference by a wide variety of modifications of hemoglobin that result in similar changes in charge.
  • Methods include:
    • Ion-exchange chromatography (high performance liquid chromatography; HPLC):
      • Cation-exchange chromatography is a process that allows the separation of the mixture based on the charge properties of the molecules in the mixture.
      • Charged hemoglobins and other hemoglobin components are eluted at varying times depending upon the net charge of the molecule in relation to a gradient of increasing ionic strength passed through a cation-exchange column.
      • The major disadvantage of cation-exchange HPLC is the interference with some hemoglobin variants.
    • Electrophoresis-based assays
      • Include:
        • Citrate agar
        • Isoelectric focusing:
          • Isoelectric focusing (IEF) in polyacrylamide gel (PAC) is a special type of electrophoresis that separates hemoglobins according to their isoelectric point.
          • A hemolysate is subjected to an electric current in a stationary pH gradient. Each hemoglobin component “focuses” as a single band in the gel at its PI.
          • The pH gradient is established by means of specialized carrier ampholytes which are most often a mixture of polyamino-polycarboxylic acids with different PIS.
      • Rarely used anymore in routine clinical laboratory settings.
• Structural differences:
  • Immunoassays:
    • Measure HbA1c specifically; antibodies recognize the structure of the N-terminal glycated amino acids (usually the first 4–10 amino acids) of the Hb β chain.
  • Assays based on boronate affinity chromatography:
    • Affinity chromatography using immobilized m-phenyl boronic acid separates glycosylated and nonglycosylated hemoglobins based on the formation of complexes between the immobilized phenylboronic acid and cis-diols in glucose-modified.

Available assays may quantify:

• All glycosylated Hb species regardless of the composition of the hemoglobin tetramer. In these methods results are usually expressed as “total gHb”.
• Products formed by glycosylation of the amino terminus of HbA (alpha 2, beta 2).
• total (HbA1a + HbA1b + HbA1c) which is often called HbA1, or total “fast” hemoglobin.
• Specific for one product, i.e., HbA1c.

In 1995, the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) convened a working group with the goal of developing a true reference method for A1c. IFCC results are reported using the International System
of Units (SI), and are expressed as millimoles of A1c per mole of HbA.

In 1996, the National Glycohemoglobin Standardization Program (NGSP) was created to standardize A1c results to ensure consistency and comparability across a variety of clinical laboratories, allowing patients with diabetes to be treated to standardized targets. NGSP-derived A1c results are reported as a percent. A1c is typically reported as an NGSP-derived percent in the United States and Canada.

Over the past several years, there has been expanded use of point-of-care (POC) assays to measure A1c. Advantages of POC testing include:

• Fingerstick sampling in the provider’s office
• Immediate patient and provider feedback leading to
• Timely adjustments in the treatment regimen

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Non-glycemic variables affecting HbA1c levels:

There are a number of factors, including analytical and biological that may affect HbA1c levels. Falsely high or low HbA1c reading can lead to overtreatment or undertreatment of diabetes. If a patient’s A1C test results are at odds with their blood glucose testing results, interference should be considered. As stated by the ADA: Notable differences between HbA1c and plasma glucose levels should raise suspicion of HbA1c assay interference; consider using an assay without interference or plasma blood glucose criteria to diagnose diabetes.

What the sources say:

Falsely elevated A1C:

• HbA1c may be increased by a number of factors:
  • Conditions that prolong the life of the erythrocyte or is associated with decreased red cell turnover exposes the cell to glucose for a longer period of time, resulting in higher A1c levels, regardless of the assay method used:
    • Asplenia
    • Iron deficiency
      • Prolongation of RBC lifespan because new RBC production in is impaired.
      • Malondialdehyde levels, increased in IDA, may increase rate of hemoglobin glycation.
    • Folate and vitamin B12 deficiency
      • Impaired production of new RBCs and increased life span of existing RBCs.
    • Late pregnancy in nondiabetic individuals
      • Possibly owing to iron deficiency
  • Conditions that interfere with assay
    • Severe hypertriglyceridemia (concentrations >1,750 mg/dL)
    • Severe hyperbilirubinemia (concentrations >20 mg/dL)
    • Bilirubin migrates with the fast Hb on charge separation method
    • Uremia:
      • Chemically modified derivatives of hemoglobin (e.g. carbamylated Hb in patients with renal failure)
      • Severe chronic kidney disease may increase RBC glycation through lipid peroxidation of hemoglobin and by extending erythrocyte life span due to decreased erythropoietin levels, causing false elevation of the HbA1c level.
      • KDIGO recommends: (i) not treating to an HbA1c target of <7.0% in patients at risk of hypoglycemia (advanced CKD patients were considered to be included in this group), and (ii) that target HbA1c be extended above 7.0% in individuals with comorbidities or limited life expectancy and risk of hypoglycemia.
      • KDIGO also assumes that HbA1c may not be reflective of glucose control in people with CKD and should be interpreted with caution, suggesting that daily self-monitoring of blood sugar levels may be more reliable.
    • Medications and substances:
      • Lead poisoning
      • Chronic ingestion of alcohol
      • Salicylates
      • Opioids
      • Ingestion of vitamin C (when measured by electrophoresis)
    • Hemoglobin variants (see section below)

Falsely lowered A1C:

• HbA1c may be increased by a number of factors:
  • Conditions associated with shorter RBC survival result in less time for hemoglobin to be exposed to glycation, reducing HbA1c concentration, regardless of the assay method used:
    • Hemolysis
    • Acute blood loss
    • Splenomegaly
  • End-stage renal disease – primarily due to the associated chronic anemia with decreased red cell survival
  • Pregnancy:
    • A1c values decline during pregnancy by 12–16 weeks of gestation with a further decrease that plateaus by gestational weeks 20–24.
    • Mechanisms include:
      • Decreased life span of the red blood cell from about 120 days to about 90 days
      • Increased erythropoietin production
    • Because A1c values are generally falsely low during pregnancy, A1c should not be used for diagnosing gestational diabetes.
• Hemoglobin variants (see section below)

Note: In summary, when evaluating a patient with diabetes and one of the conditions described above (most common are renal failure and pregnancy), the clinician should consider that the A1c result may be falsely lowered and not reflective of the patient’s true level of average glycemia.

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Hemoglobin variants and HbA1c levels:

Certain hemoglobin traits, such as HbS, HbC, HbF, and HbE, interfere with some A1C assay methods. By using methods that separate by migration based on molecular charge, a hemoglobin variant molecule can migrate similarly to HbA1c and create a falsely elevated or depressed reading. This situation produces an artifact whereby the hemoglobin variant is being measured instead of or in addition to HbA1c. In people who have hemoglobin variants such as HbS (sickle cell trait), some A1C tests give falsely high or low readings that can lead to the over-treatment or under-treatment of diabetes. Currently, many assay methods can correct for the presence of the most common hemoglobin traits and reliable A1C tests are available for people with most hemoglobin variants.

In most current HbA1c assays, hemoglobin variants prevalent in SCD patients such as HbS or HbF do not cause interference. However, HbA1c assays can’t account for the difference in RBC survival that occurs with hemoglobinopathies. Further compounding this issue is evidence suggesting that at a given glucose concentration, white individuals actually have overall lower HbA1c levels than those of African descent. Other factors related to SCD/SCT such as iron deficiency anemia can also affect HbA1c measurements.

Health care professionals should suspect the presence of a hemoglobinopathy when:

• An A1C result is unexpected or at odds with other diabetes test results.
• An A1C result is below 4 percent or above 15 percent.
• The results of self-monitoring of blood glucose differ from A1C results.
• A patient’s A1C result is significantly different from a previous A1C result, following a change in laboratory A1C methods.

Practice point (NIDDK):¹

Health care professionals or patients interested in the accuracy of a particular A1C method for patients with hemoglobin variants should first find out which method their laboratory is using. With some assay methods, A1C tests in patients with hemoglobinopathies result in falsely high outcomes, overestimating actual average blood glucose levels for the previous 3 months. Health care professionals might falsely diagnose patients or prescribe more aggressive treatments, resulting in increased episodes of hypoglycemia. Some assay methods used with certain hemoglobinopathies may result in falsely low outcomes, leading to undertreatment of diabetes. Health care professionals should not use the A1C test for patients with HbSS, HbCC, or HbSC. These patients may suffer from anemia, increased red blood cell turnover, hemolysis, and transfusion requirements, which can adversely affect A1C as a marker of long-term glycemic control.

A note about assays based on charge differences (e.g., ion exchange chromatography (HPLC) assays):

• Change in amino acid in the most common Hb variants (S, C E, D) causes a change in the net charge that affects measurement of HbA1c by methods based on charge difference such as ion exchange chromatography.
• Leads to a situation where Hb variants, both glycated and non-glycated, co-elute or co-migrate with HbA1c, causing an overestimate of the latter.

A note about immunoassay methods:

• Because the S and C variants are close to the N terminus on the β chain, some (but not all) immunoassays are affected by the presence of these variants.
• The presence of HbE or HbD, however, with mutations much further away on the β chain, generally does not affect immunoassay methods.

A note about assays based on boronate affinity chromatography:

HbS, C, E, and D affect the ionic charge of the Hb molecule, which may cause interference with ion-exchange methods, depending on how well the variant Hb is separated from HbA. Since boronate affinity chromatography separates total glycated hemoglobin from nonglycated hemoglobin, regardless of the hemoglobin species, there is generally no interference from most Hb variants.

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