HbA1c Testing in the laboratory.

Summary

  • HBA1c is a blood test used in monitoring glycemia in patients with diabetes mellitus
  • Hemoglobin forms non-covalent bonding with glucose forming glycated hemoglobin
  • The level of HBA1c is correlated with the level of glucose in the blood for a preceding  period of 2-3months
  • Measuring HBA1c gives an indication of how well blood glucose has been in the past 2-3 months.
  • Levels of HBA1c are closely correlated with the risk of long-term complications of diabetes mellitus.
  • The target value for HBA1c for Kenya is <6.5% and is based on WHO guidelines.
  • Target glycemic control should be individualized for each patient.

Principle of the HbA1c Test:

This method uses TTAB* as the detergent in the hemolyzing reagent to eliminate interference from leukocytes (TTAB does not lyse leukocytes). Sample pretreatment to remove labile HbA1c is not necessary.
All hemoglobin variants which are glycated at the *Beta-chain N-terminus and which have antibody recognizable regions identical to that of HbA1c are measured by this assay. Consequently, the metabolic state of patients having uremia or the most frequent hemoglobinopathies (HbAS, HbAC, HbAE) can be determined using this assay.

*TTAB = Tetradecyltrimethylammonium bromide

Hemoglobin

Liberated hemoglobin in the hemolyzed sample is converted to a derivative having a characteristic absorption spectrum which is measured bichromatically during the pre-incubation phase (sample + R1) of the above immunological reaction. A separate Hb reagent is consequently not necessary. 

Hemoglobin A1C

The HbA1c determination is based on the turbidimetric inhibition immunoassay (TINIA) for hemolyzed whole blood.

  1. Sample and addition of R1 (Antibody reagent): Glycohemoglobin (HbA1c) in the sample reacts with anti-HbA1c antibody to form soluble antigen-antibody complexes. Since the specific HbA1c antibody site is present only once on the HbA1c molecule, complex formation does not take place.
  2. Addition of R3 (Polyhapten reagent) and the start of reaction: The polyhaptens react with excess anti-HbA1c antibodies to form an insoluble antibody-polyhapten complex which can be measured turbidimetrically

Clinical significance:

Diabetes causes elevated levels of glucose to circulate in the blood. Maintaining normal levels of blood glucose is part of the routine clinical management of diabetes. Continuous and careful management of blood glucose levels prevents development of serious long-term complications resulting from vascular impairment such as retinopathy, nephropathy and neuropathy.


Although a fasting blood glucose measurement gives the clinician information about the patient’s status over the last twelve hours, the stable HbA1c offers a more accurate indication of the patient’s long-term diabetic control over the last two to three months.
Hemoglobin (Hb) consists of four protein subunits, each containing a heme moiety, and is the red-pigmented protein located in the erythrocytes. Its main function is to transport oxygen and carbon dioxide in the blood. Each Hb molecule is able to bind four oxygen molecules. Hb consists of a variety of subfractions and derivatives. Among this heterogeneous group of hemoglobins HbA1c is one of the glycated hemoglobins, a subfraction formed by the attachment of various sugars to the Hb molecule.

HbA1c is formed in two steps by the non-enzymatic reaction of glucose with the N-terminal amino group of the beta-chain of normal adult Hb (HbA)

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The first step is reversible and yields labile HbA1c. This is rearranged to form stable HbA1c in a second reaction step. In the erythrocytes, the relative amount of HbA converted to stable HbA1c increases with the average concentration of glucose in the blood. The conversion to stable HbA1c is limited by the erythrocyte’s life span of approximately 100 to 120 days. As a result, HbA1c reflects the average blood glucose level during the preceding 2 to 3 months. HbA1c is thus suitable to monitor long-term blood glucose control in individuals with diabetes mellitus. Glucose levels closer to the time of the assay have a greater influence on the HbA1c level.

The risk of diabetic complications, such as diabetic nephropathy and retinopathy, increases with poor metabolic control. In accordance with its function as an indicator of the mean blood glucose level, HbA1c predicts the development of diabetic complications in diabetes patients. For routine clinical use, testing every 3 to 4 months is generally sufficient. In certain clinical situations, such as gestational diabetes, or after a major change in therapy, it may be useful to measure HbA1c in 2 to 4-week intervals.

HbA1c Testing Recommendations

  1. Perform the A1C test at least two times a year in patients who are meeting treatment goals (and who have stable glycemic control).  
  2. Perform the A1C test quarterly in patients whose therapy has changed or who are not meeting glycemic goals. 
  3. Point-of-care testing for A1C provides the opportunity for more timely treatment changes. 

Ref: Glycemic Targets: Standards of Medical Care in Diabetes—2019 American Diabetes Association Diabetes Care Jan 2019, 42 (Supplement 1) S61-S70; DOI: 10.2337/dc19-S006

HbA1c Specimen collection and Processing:

  • Universal Precautions apply.
  • Collect whole blood specimens in vacuum collection tubes containing EDTA and mix thoroughly. 
  • The minimum volume required for analysis directly from collection tubes is 1 mL of whole blood. Whole blood samples as small as 100 uL may be run in sample cups – only for QC, patients must be in a tube.

Specimen Storage and Stability:

  • 3 days at 15-25 °C
  • 7 days at 2-8 °C
  • 6 months at (-15) – (-25) °C

Calculation

The final result is expressed as percent HbA1c and is calculated from the HbA1c/Hb ratio as follows: Protocol 2 (acc. To DCCT/NGSP): HbA1c (%) = (HbA1c/Hb) x 91.5 + 2.15

The following equation is used to calculate the eAG (mg/dL): eAG (mg/dL) = (28.7 x HbA1c) – 46.7

Interpretation

The reference value for  HbA1c is <6.5%.
This is the generalized value used in kenya based on the WHO guidelines. Of note is that HBA1c is used in the context of glycemic control monitoring and not in the diagnosis of diabetes mellitus.

Based on the results of the A1c-Derived Daily Glucose (ADAG) study, the ADA recommends the use of a new term in diabetes management, estimated average glucose, or eAG. This new calculation is intended to help health care providers report HbA1c results to patients using the same units that patients see routinely in blood glucose measurements.

Correlation of HBA1c with glucose levels

Considerations for Glycemic controls

HBA1c levels are well correlated with the risk of long-term complications of diabetes. It is therefore paramount to achieve the best glycemic controls possible. In reality, a myriad of factors determines what is feasible for each patient. The infographic below gives some of the considerations when setting targets with individual patients.

Limitations of the HbA1c Test

  1. For diagnostic purposes, mmol/mol HbA1c values (IFCC) and % HbA1c values (DCCT/NGSP) should be used in conjunction with information from other diagnostic procedures and clinical evaluations.
  2. The test is designed only for accurate and precise measurement of mmol/mol HbA1c (IFCC) and % HbA1c (DCCT/NGSP). The individual results for total Hb and HbA1c concentration should not be reported.
  3. The test is not intended for the diagnosis of diabetes mellitus or for judging day-to-day glucose control and should not be used to replace daily home testing of urine or blood glucose.
  4. As a matter of principle, care must be taken when interpreting any HbA1c result from patients with Hb variants. Abnormal hemoglobins might affect the half-life of the red cells or the in vivo glycation rates. In these cases even analytically correct results do not reflect the same level of glycemic control that would be expected in patients with normal hemoglobin.
  5. Any cause of shortened erythrocyte survival will reduce exposure of erythrocytes to glucose with a consequent decrease in mmol/mol HbA1c values (IFCC) and % HbA1c values (DCCT/NGSP), even though the time-averaged blood glucose level may be elevated. Causes of shortened erythrocyte lifetime include:-
    •  hemolytic anemia or other hemolytic diseases
    • homozygous sickle cell trait 
    • pregnancy, 
    • recent significant or chronic blood loss, etc.
  6. Glycated HbF is not detected by the assay as it does not contain the glycated *Beta-chain that characterizes HbA1c. However, HbF is measured in the Total Hb assay and as a consequence, specimens containing high amounts of HbF (> 10 %) may result in lower than expected mmol/mol HbA1c values (IFCC) and % HbA1c values (DCCT/NGSP).

How to get your HBA1C Test Conveniently and Affordably.

You can order your HBA1C test easily from your phone or computer. Simply follow this link to order your test online. No doctor appointment or insurance is required.

Additional professional resources.

Download a free editable version of the HbA1c Standard Operating Procedure(SOP) that you can adapt and use in your lab here.
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