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.