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Hemoglobin A1c

Hemoglobin A1c (HbA1c) refers to a minor population of HbA that has been modified by attachment of glucose to the N-terminal amino acid of the beta globin chain. Since erythrocytes are freely permeable to glucose, the attachment occurs continually over the entire lifespan of the erythrocyte and is dependent on glucose concentration and the duration of exposure of the erythrocyte to blood glucose.

Hemoglobin A1C

 

HbA1c is a weighted average of blood glucose levels during the preceding 120 days, which is the average life span of red blood cells. A large change in mean blood glucose can increase HbA1c levels within 1-2 weeks. Sudden changes in HbA1c may occur because recent changes in blood glucose levels contribute relatively more to the final HbA1c levels than earlier events. For instance, mean blood glucose levels in the 30 days immediately preceding blood sampling contribute 50% to the HbA1c level, whereas glucose levels in the preceding 90-120 day period contribute only 10%. Thus, it does not take 120 days to detect a clinically meaningful change in HbA1c following a significant change in mean plasma glucose level.

Hemoglobin A1c Methods

Methods for analysis of HbA1c can essentially be divided into 2 categories depending on whether they measure HbA1c based upon charge or structure. The most common charge-based method utilizes cation-exchange high pressure liquid chromatography (HPLC). In this method, different hemoglobin molecules (eg, HbA, HbA2, HbF) are eluted from the column at different times following exposure to buffers of increasing ionic strength, depending on their charge. HbA1c is less positively charged than HbA and does not bind as tightly to the negatively charged resin. Therefore, it elutes more rapidly than HbA. The quantity of each Hb fraction in the eluate is quantitated by spectrophotometry and expressed as a percentage.

Structural methods include boronate-affinity chromatography and immunoassays. Boronate-affinity chromatography is based on the strong bindng of the coplanar cis-diol groups on glycated hemoglobin with boronic acid residues attached to the chromatography resin. Nonglycated Hb does not bind to boronic acid and elute immediately. Glycated Hb bind to boronic acid and must be eluted by a counterligand. This method measures total glycated hemoglobin, including HbA1c and Hb glycated at the epsilon amino groups on lysine. It must be calibrated using HbA1c specific values to produce HbA1c equivalent values.

Most immunoassays use polyclonal or monoclonal antibodies that recognize the β-N-terminal glycated amino acid in the first 4 to 10 amino acids of beta globin. The final concentration is calculated as a ratio of HbA1c to total hemoglobin.

Hemoglobin Variants

More than 950 different hemoglobin variants have been identified. In the United States, hemoglobin S is the most common variant, followed by hemoglobin C, hemoglobin E, and hemoglobin D (Punjab/Los Angeles). Worldwide, hemoglobin variants follow this same trend, except that hemoglobin E is more common than C. Hemoglobin variants that produce changes in the charge or structure of the hemoglobin molecule may affect the accuracy of HbA1c measurement. In general, charge-based methods are more susceptible to interference from hemoglobin variants than immunoassays. Boronate-affinity chromatography is the least affected by hemoglobin variants. Situations that may suggest interference with HbA1c measurement due to a hemoglobin variant include:

  • Poor correlation of self-blood glucose monitoring with A1C results
  • A HbA1c result different than expected
  • A HbA1c result greater than 15%
  • A HbA1C result differing significantly from a previous result obtained with a different method

The following table lists the methods most often used to measure A1C and whether the method is affected by HbS or HbC or HbE or HbD trait.

Interference from

Method

HbAS

HbAC

HbAE

HbAD

Abbott Architect

Yes

Yes

-

-

Axis-Shield Afinion

No

No

No

No

Bayer A1cNOW

Yes

Yes

No

No

Beckman Synchron

No

No

No

No

Bio-Rad D-10

No

No

No

No

Bio-Rad Variant A1c

No

No

No

Yes

Bio-Rad Variant II A1c

No

No

No

No

Bio-Rad Variant II Turbo A1c

No

No

Yes

Yes

Dade Dimension

No

No

No

No

Olympus AU

Yes

Yes

No

No

Ortho-Clinical Vitros

No

No

No

No

Primus HPLC (affinity)

No

No

No

No

Roche Cobas Integra Gen.2

No

No

No

No

Roche/Hitachi (Tina Quant II)

No

No

No

No

Siemens (Bayer) Advia A1c

No

No

-

-

Siemens (Bayer) DCA 2000

No

No

-

-

Tosoh A1c 2.2 Plus

No

No

Yes

No

Tosoh G7

No

No

Yes

No

Tosoh G8

No

No

Yes

No

Modified from http://www.ngsp.org/interf.asp

If a patient has one of these hemoglobin variants, their HbA1c level should be measured using a method that does not show interference from the variant. Patients with serious hemoglobinopathies (such as homozygous S or C) may have inaccurate HbA1c levels due to shortened red cell lifespan. In these cases, an alternative test of glycemia, such as fructosamine (also called glycated serum protein or glycated albumin) may be substituted. Serum proteins show average glucose levels over a much shorter period of time than the A1C test, usually about 2 to 3 weeks. Moreover, the fructosamine test has not been standardized and the relationship of results of this test to glucose levels or risk for complications has not been established

Conditions that affect HbA1c Level

Hemodilution and increased red cell turnover decrease HbA1c concentration during pregnancy. Iron deficiency can increase HbA1c level during the last trimester due to decreased cell turnover.

Hemolytic anemia and blood loss anemia decrease HbA1c level due to the release of reticulocytes whose hemoglobin is not glycosylated. Transfusion of diabetic patients with red blood cells decreases their HbA1c concentration. Patients with asplenia have higher HbA1c levels due to increased circulating life span of red blood cells. Iron deficiency increases HbA1c, but iron replacement therapy increases reticulocytosis and decreases HbA1c concentration. Vitamin B12 deficiency also decreases red cell turnover and increase HbA1c level.

Patients with renal failure have misleadingly low HbA1c levels due to shortened red cell survival and the use of recombinant erythropoietin, which increases reticulocytosis.

Chronic alcohol, salicylate and opiate use have all been reported to falsely increase HbA1c levels. Drugs that cause hemolysis can lower HbA1c concentration. Examples include dapsone, ribavirin and sulfonamides. Hydroxyurea converts hemoglobin A to F, thereby lowering HbA1c.

Clinical Use

HbA1c provides a much better indication of long term glycemic control than blood and urinary glucose determinations. There is broad consensus that HbA1c levels should be used for routine care of all patients with diabetes mellitus. Baseline HbA1c levels are strongly related to the incidence and/or progression of retinopathy, gross proteinuria, and loss of tactile sensation or temperature sensitivity. In the 2009 American Diabetes Association (ADA) Summary of Revisions for the Clinical Practice Recommendations, the HbA1c goal for nonpregnant adults is <7% (Diabetes Care 2009;32(S1):S3-S5).

Currently, the American Diabetes Association (ADA) advocates that all individuals at high risk for diabetes be screened at least every 3 years. All persons 45 years and older are considered to be at high risk. ADA also recommends measurement of HbA1c three to four times per year for type 1 and poorly controlled type 2 diabetic patients, and 2 times per year for well-controlled type 2 diabetic patients. This is a general guideline to reduce the risk of microvascular and neuropathic complications. Physicians may need to individualize testing for certain patients.

The Office of Inspector General (OIG) has released its Fiscal Year 2011 Work Plan, which describes the investigative, enforcement and compliance activities that it will undertake in the coming year.

OIG will review Medicare contractors for screening the frequency of clinical laboratory claims for HbA1c and determine the appropriateness of Medicare payments. The following testing intervals are considered medically necessary:

  • Every 3 months to monitor a diabetic patient’s metabolic control
  • Every 1-2 months when treatment regimen is altered to improve control
  • Every month for diabetic pregnant women
  • Patients with uncontrolled type I or II diabetes may be tested more frequently if the medical record contains supportive documentation

An Advance Beneficiary Notice (ABN) should be submitted for Medicare patients whenever a HbA1c is ordered at more frequent intervals than those listed above.

HbA1c for Diagnosis of Diabetes

Hyperglycemia has been the sole diagnostic criterion for diabetes since the development of blood glucose assays 100 years ago. Despite being the gold standard, measurement of blood glucose is less accurate and precise than most physicians realize. Blood glucose measurements are also subject to several limitations including:

  • 8-12 hour fasting specimen requirement
  • Diurnal variation requiring morning collection to capture peak levels
  • Large biological variation of 5-8%
  • Nonstandardized instrument methods with >12% bias
  • Glycolysis after collection, even in sodium fluoride tubes

In 2009, an International Expert Committee recommended the use of the HbA1c test to diagnose diabetes, with a threshold of 6.5% or greater (Diabetes Care 2009, 32 (7):1327-1334). The American Diabetes Association adopted this criterion in 2010. The diagnostic cutpoint of 6.5% was recommended based on the risk for developing microvascular complications such as retinopathy. This HbA1c criterion identifies one third fewer cases of undiagnosed diabetes than a fasting glucose cut point of 126 mg/dL or greater. However, the advantages of using HbA1c outweigh this limitation.

  • Better index of overall glycemic exposure & risk of complications
  • Low intraindividual variability (<2%)
  • No requirement for fasting or timed specimen
  • Standardized methods with precision <2%
  • Less affected by acute illness or stress
  • Good stability after blood collection
  • Single test can be used for both diagnosis and monitoring

Patients who have an HbA1c of 5.7 to 6.4% are considered at high risk for developing diabetes and cardiovascular disease in the future. These individuals are referred to as having prediabetes. They should be identified and counseled about lifestyle modifications such as exercise and weight loss.

An elevated HbA1c should be confirmed with a repeat measurement on a different day, except in those individuals who are symptomatic and also have plasma glucose over 200 mg/dL. HbA1c testing is indicated in children in whom diabetes is suspected but the classic symptoms and a casual glucose >200 mg/dL are not found. Analysis should be performed on central laboratory instruments and not with point of care devices, which have not been shown to be sufficiently accurate or precise for diagnosis.

Test

Non-diabetes

Prediabetes

Diabetes

HbA1c

4.0 – 5.6%

5.7 – 6.4%

6.5% or >

Fasting plasma glucose

<100 mg/dL

100 – 125 mg/dL

126 mg/dL or >

2 hour glucose

<140 mg/dL

140 – 199 mg/dL

200 mg/dL or >

 

Estimated Average Glucose

Physicians often correlate the HbA1c level with a patient’s glucose meter history and fasting plasma glucose concentrations. The Diabetes Control and Complications Trial (DCCT) clearly demonstrated a direct relationship between HbA1c and mean plasma glucose levels (Diabetes Care 2002; 25:275-78). Thus, HbA1c can be used to calculate the estimated average glucose (eAG). The following table summarizes the relationship.

HbA1c (%)

eAG (mg/dL)

4

65

5

97

6

126

7

154

8

183

9

212

10

240

11

269

12

298

 

Average glucose concentration is calculated from HbA1c concentration using the formula: eAG = (28.7 x %HbA1c) – 46.7. Each 1% change in HbA1c represents a change of approximately 30 mg/dl in plasma glucose. It is important to realize that this data is based on overall averages and may vary in individual patients. Reporting of the eAG at this time is still controversial and is not routinely offered by clinical laboratories at this time.

Hemoglobin A1c as a CV Risk Factor in Nondiabetic Individuals

Macrovascular disease is the most important cause of mortality and morbidity in individuals with type 2 diabetes. Even when adjusted for conventional risk factors, diabetic individuals still exhibit a two to four fold increased risk of cardiovascular disease in comparison to nondiabetic people. Therefore, hyperglycemia is strongly suspected of promoting atherogenesis. Excess glucose is transformed into advanced glycation endproducts (AGEs) that not only make blood vessels inelastic and stenotic but also activates chronic inflammation.

Recent studies have demonstrated that HbA1c is also a predictor of all-cause, cardiovascular and ischemic heart disease mortality even at concentrations below the accepted threshold for diabetes (British Med J 2001; 322:15-18). The following table lists the relative risk of death for each quartile of HbA1c concentration.

Mortality

<5%

5.0 – 5.4%

5.5 – 6.9%

7% or >

All Cause

1.0

1.41

2.07

2.64

CV

1.0

2.53

2.46

5.04

Ischemic

1.0

2.74

2.77

5.20

 

Individuals with HbA1c concentrations above 5% have greater risk than individuals with concentrations below 5%. Approximately 25% of population has HbA1c levels below 5% and 70% of the population had levels between 5 and 6.9%. HbA1c appears to resemble blood pressure and cholesterol in terms of its continuous relationship with cardiovascular risk.

Two recent studies in the Annals of Internal Medicine have also validated that HbA1c is a progressive risk factor for CV disease in individuals with and without diabetes (Ann Intern Med 2004; 141:413-20 & 421-31). Every 1% absolute increase in HbA1c above the nonglycemic level of 5% predicts a 20% relative increase in the incidence of CV events even after adjustment for systolic blood pressure, cholesterol level, body mass index, waist to hip ratio, smoking and previous myocardial infarction or stroke. A similar relationship exists for total mortality.

Specimen requirement is one lavender top (EDTA) tube of blood.

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