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HbA1c and Assay Methods
What are Glycated Hemoglobins?
Glycated hemoglobins arise from the non-enzymatic attachment of glucose to hemoglobin. They are formed and accumulate in the red cell in proportion to the blood glucose level. Their concentration reflects the long-term average glucose level and is thus useful as an indicator of diabetic control. The formation of HbA1c proceeds via a Schiff base adduct, or aldimine, followed by the Amadori rearrangement to form the stable ketoamine, HbA1c. The aldimine intermediate is often referred to as labile HbA1c and the rate of its conversion to the ketoamine is some 60 times slower than the reverse dissociation with release of the glucose.
What is Hemoglobin?
Hemoglobin is the red blood protein that transports oxygen from the lungs to the tissues. It is the major component of the red cell. Because the life of the red cell averages some 120 days, the extent that the hemoglobin is glycated can be used as an indication of the average glucose level over the previous one or two months.
A molecule of hemoglobin is made up of 4 protein chains. There are 2 alpha chains and 2 beta chains, usually denoted as α2β2. Glucose will react and bond to certain positively charged chemical groups on the hemoglobin. These are located at the start (N-terminal end) of each of the α and β chains and on some amino acid side chains within the protein. In particular glucose reacts with the ε-amino group of lysine amino acids.
What is the difference between HbA1c and Glycated Hemoglobin?
Glycated hemoglobin is defined as hemoglobin with glucose bound to any of these potential sites. HbA1c is a subset of glycated hemoglobins. It is defined as hemoglobin with glucose bound at the beginning (N-terminal) of the β-chain. The total glycated hemoglobin will include HbA1c plus all the other hemoglobins that have glucose bound to lysine side chains and/or to the N-terminal of the α-chain. Generally about half of the glucose is bound to the HbA1c position with the other half bound at 3 or 4 other sites (lysines).
How is HbA1c Measured?
The concentration of HbA1c is measured and expressed as a percentage of the total hemoglobin. The methods used to analyze HbA1c fall into one of three main types. These are:
- Ion Exchange HPLC
- Immuno Assay
- Glycation specific. This includes boronate affinity and chemical methods such as the thiobarbituric acid method.
Analysis of HbA1c on Ion Exchange HPLC
When glucose binds at the β N-terminal to form HbA1c the hemoglobin molecule undergoes a conformational change that results in the molecule presenting with an extra negative charge. This means that on ion-exchange chromatography systems it will separate out from normal hemoglobin (HbA0) and from hemoglobin glycated at lysine side chains which often co-elute with HbA0. With refinement of chromatography methods and instrumentation (HPLC), hemoglobin is routinely separated into a number of peaks such as HbA1a, HbA1b, HbF (fetal Hemoglobin), HbA1c, HbA0, HbA2 and a number of hemoglobin products resulting from reaction with other molecules (glutathione, urea, aspirin etc). There are also a number of hemoglobin degradation products which show up with samples that have aged. On some HPLC systems these may co-elute with, or be incompletely separated from, HbA1c. In these cases, the HbA1c value obtained may be reported as higher than it actually is. Fresh blood samples contain a significant amount of labile HbA1c which in some of the earlier analyzers is removed by a pre-incubation step. Newer analyzers separate the labile form from the stable HbA1c and do not need pre-incubation of the sample.
Platforms that use ion exchange HPLC include BioRad D-10, Diastat, Variant, Variant II and Variant Turbo; TOSOH 2.2 Plus, and G7. The integrity of a HbA1c control is more critical with these methods because any degradation can result in the increase of interfering peaks that may co-elute (and co-integrate) with HbA1c. For this reason, the useable reconstituted or open vial times for these methods may be less than for an Immuno Assay procedure.
Analysis of HbA1c by Immuno Assay Procedures
A typical method uses a specific antibody (usually monoclonal) to the glucose and the first 5 to 10 amino acids of the β-chain. This antibody is latex coated. There is also an agglutinator which is a synthetic polymer with multiple copies of the HbA1c portion that the antibody targets. The agglutinator reacts with the antibody to give a scattering of light and an increase in absorbance. The HbA1c from the blood sample then competes with the agglutinator for the antibody latex binding sites and thus reduces the scattering of light and the absorbance. So the greater the HbA1c in the blood, the less the absorbance will be from scattered light. From this the amount of HbA1c is calculated, and the total hemoglobin can be determined by measuring at or near the Soret absorption band of hemoglobin (410 - 420nm) or by Drabkins method (oxidation and conversion to cyanmethemoglobin) at about 540nm.
Assay platforms using an immuno assay procedure include; Siemens Healthcare Diagnostics DCA Series and Advia 1650, Dade Behring Dimension, Roche Cobas and Hitachi. Most of the reactions on hemoglobin from aging or other chemical or free radical type reactions do not occur at the HbA1c site where the antibody in this method group is targeted. For this reason this type of method is generally more tolerant of aging/degradative reactions and HbA1c controls would be expected to have longer useable reconstituted or open vial times than ion exchange HPLC procedures.
Analysis of HbA1c by Glycation Specific Procedures
There are two main methods here. The older thiobarbituric acid method (or variants of it) is not used much now. This is a chemical method and measures the total amount of glucose bound to the hemoglobin. The results can be expressed as moles of glucose or fructosamine per mole of hemoglobin or heme and are converted to %HbA1c using a calibration graph.
The more popular method in this class is the boronate affinity procedure. This employs phenylboronate attached to an inert resin. Phenylboronate binds to cis-hydroxy groups such as are on the glucose attached to hemoglobin. The passage of blood through a boronate affinity column results in the glycated hemoglobin binding to the resin and the unglycated hemoglobin being eluted straight through. The bound glycated fraction is released from the column using a competitive boronate binding compound such as sorbitol.
The ratio of glycated to total hemoglobin is determined and this is converted to %HbA1c by multiplying by a factor determined from a calibration graph or formula. This ratio is assumed to be constant. In other words, the pattern of native (in vivo) glycation is considered to be the same for all patients and levels of glycation.
This method will give the same HbA1c result for a hemoglobin with two glucoses attached per molecule as a hemoglobin with one glucose per molecule. In contrast, the thiobarbituric acid method will measure the amount of hemoglobin with 2 attached glucose residues as being twice that of the hemoglobin with one glucose.
Assay platforms that use glycation specific methods include the Primus analyzers, the BioRad Micromat II, and the Nicocard. These assays have the advantage of not being affected by variant hemoglobins.
Research Methods
The best primary reference methods use electrospray ionization mass spectrometry to accurately measure the ratio of the β chain N-terminal hexapeptide with the glucose attached to that without the glucose.
Why Measure HbA1c?
The measurement of HbA1c is especially useful in insulin-dependent diabetic patients where blood glucose levels fluctuate widely and where the instantaneous blood glucose does not reflect the averaged situation. The formation of HbA1c occurs slowly (about 0.05%/day) and continuously during the 120-day lifetime of the red cell. Hence the measurement of HbA1c is useful to physicians as a long-term integral of blood glucose concentration and thus as a measure of the degree of control or self-management by the diabetic patient. The normal range for HbA1c is 4% - 6% of total hemoglobin. Each percentage point increase in HbA1c level corresponds to an increase in average blood glucose level of about 30 mg/dL or 1.7mmol/L. As a general rule HbA1c levels above 10% represent poor diabetic control, whereas values between 6.5% and 7.5% are indicative of good control.
The extendSURE™ Controls
Stability of Hemoglobin Controls
The stability of a hemoglobin quality control is an important issue. A good control should continue to give the same result on repeated analysis during the defined life of the control. Hence, the control must be stable not only during its life in the diagnostic laboratory but during transportation from the manufacturer or distributor to the laboratory.
Degradation of hemoglobin occurs as a result of chemical and free radical reactions. The integrity of the control can be damaged as a result of a poorly designed lyophilization protocol, or by repeated freezing and thawing, or as a result of microbial growth. So it is important during the design of a control that deleterious reactions and conditions are identified and the production and formulation of the controls is planned so that these unwanted reactions are inhibited or their rates dramatically reduced. It is also important that any protective additives do not interfere with the HbA1c analytical methods.
The Canterbury Scientific Ltd controls have a careful formulation that include a number of protectants and stabilizers that preserves the hemoglobin in its native conformation. This results in a product with exceptional stability.
In Vitro Glycation
Canterbury Scientific Ltd produces the abnormal level (Level 2) HbA1c control by a process of controlled in vitro glycation of non-diabetic blood. This means there is not a reliance on sourcing blood from diabetic subjects with high HbA1c levels, and it also means that levels of up to over 20% can be produced if required. It also avoids the potential ethical issues relating to obtaining blood from a patient with a clinical condition.
In vitro glycation essentially glycates the same amino acid residues as are glycated in the native glycated hemoglobin. But the ratio of α:β chain glycation is higher in the in vitro product. This means that there is an increase in the proportion of glycation at sites other than the HbA1c site. This makes no difference in procedures such as ion exchange HPLC, or immuno assay where the analytical method specifically measures the HbA1c directly (i.e. measures the glycation of the β N-terminal valine). However it means that with glycation specific type assays such as the boronate affinity method the calculated HbA1c figure (with the increased proportion of glycation at sites other than the HbA1c site) will be higher than when the same glycated sample is measured on the 2 other method types. The normal level control is unaffected since it has been glycated in vivo.
Lyophilized Hemoglobin A1c Control
The lyophilized Hemoglobin A1c control is a two level control with defined levels between 4.5% - 6.0% (Normal, Level 1) and between 9.0% - 14.0% (Abnormal, Level 2). They are intended as a quality control material for analytical methods including ion exchange HPLC, latex immunoagglutination inhibition and boronate affinity for quantitation of HbA1c. The controls are in glass vials with screw top caps.
The source blood is tested as non-reactive for Hepatitis B surface antigen, Hepatitis C antibody, antibodies against human immunodeficiency virus (HIV) types 1 & 2, HIV-1 antigen, and Syphilis (RPR or TPHA).
The standard product has a reconstituted volume of 0.25mL with a total hemoglobin of 14g/dL and is supplied in a kitset containing 2 normal and 2 abnormal level controls, 1 vial of reconstitution fluid, 4 glass droppers and a control information sheet. It is also available in a mini bulk pack containing 12 vials of Normal and Abnormal level with 4 vials of reconstitution fluid, or in a bulk pack of 100 vials each of normal and abnormal level and 50 vials of reconstitution fluid. Canterbury Scientific will also produce special orders with different vial fills and/or kitset contents.
The lyophilized product is stable for 3 years from manufacture with 3 months for the reconstituted control at 2° to 8°C.
Liquid Stable Hemoglobin A1c Control
The Liquid Stable Hemoglobin A1c control is a two level diabetic control. Level 1 has an HbA1c with a defined value within the normal range of 4.5% - 6.0%. In Level 2 the HbA1c has a defined value between 9.0% - 14.0%. They are intended as a quality control material for analytical methods including ion exchange HPLC, latex immunoagglutination inhibition and boronate affinity for quantitation of HbA1c. The liquid controls are in vials with screw top caps.
They are prepared from normal non-diabetic adult human blood. The source blood is tested as non-reactive for Hepatitis B surface antigen, Hepatitis C antibody, antibodies against human immunodeficiency virus (HIV) types 1 & 2, HIV-1 antigen, and Syphilis (RPR or TPHA).
The controls are available in 0.5mL or 1.0mL per vial with a standard kitset comprising 2 normal and 2 abnormal level controls together with a control information sheet. A mini bulk pack containing 12 vials of each level, and a bulk pack of 100 vials of each level also is available.
The closed vials when stored at 2° to 8°C have an expiry date of 30 months from production. The vial once opened is stable for 30 days stored at 2° to 8°C. These stability claims are very conservative.
