TEST FOR RECENT GLYCAEMIC CONTROL: HbA1c measurement

MEASUREMENT OF GLYCATED HEMOGLOBIN

Glycation is the non enzymatic addition of sugar residue to amino groups of proteins. In adults HbA constitute the major fraction (97%) also has other subforms namely A_1a, A_1b, A_1c which are collectively called HbA₁, fast hemoglobins, glycohemoglobins or Glycated hemoglobins. HbA_1c is formed by the condensation of glucose with N-terminal valine residue of each β-chain of HbA to form an unstable Schiff base (aldimine, pre-HbA_1c). The Schiff base may either dissociate or undergo an Amadori rearrangement to form a stable ketoamine, HbA_1c. HbA_{1a1, 1a2} which make up HbA_1a have fructose-1, 6-diphosphate and glucose-6-phosphate, respectively attached to amino terminal of the β-chain. Other are HbA_1b has pyruvate attached to N-terminal of beta chain.  HbA_1c is the major fraction constituting approximately 80% of HbA₁.

Glycation may also occur at sites other than the end of beta chain, such as lysine residue or the alpha chain. These GHbs referred to as Glycated HbA₀ or total Glycated Hb. These are measured by boronate affinity chromatography.

(Source: Tietz Clinical Chemistry, 4th Edition)

Formation of GHb is essentially irreversible and the concentration in the blood depends on both the lifespan of the red blood cell (average 120 days) and the blood glucose concentration. Since erythrocyte is free permeable to glucose. Because the rate of formation of GHb is directly proportional to the concentration of glucose in the blood, the GHb concentration represents the integrated values for glucose over the preceding 6 to 8 weeks. This provides an additional criterion for assessing glucose control because GHb values are free of day to day glucose fluctuations and are unaffected by recent exercise or food ingestion.

The interpretation of GHb depends on the red blood cells having a normal lifespan. Patients with hemolytic disease or other conditions with shortened red blood cells survival exhibit a substantial reduction in GHb. Similarly individuals with recent significant blood loss have false low values owing to higher fraction of young erythrocytes. High GHb concentrations have been reported in iron deficiency anemia, probably because of high proportion of old erythrocytes. Presence of other hemoglobinopathies can alter results. Presence of carbamylated Hb which is formed by attachment of urea and is present in large amount in renal failure and common in diabetic patients, also produce altered results.

GHb has been established as an index of long term blood glucose concentration and as a measure of the risk for the development of complications in patients with diabetes mellitus. There is direct relationship between blood glucose concentration (assessed by HbA_1c) and the risk of complications. The absolute risks of retinopathy and nephropathy were directly proportional to the mean HbA_1c. Studies have shown reduction in HbA_1c level will significantly reduce the risk of microvascular complications and retinopathy and nephropathy and cardiovascular disease. ADA recommends that a primary treatment goal in adults with diabetes should be near normal glycemia with HbA_1c <7%. HbA1c of 7% (of total HbA) corresponds with mean plasma glucose of approximately 170 mg/dl, and each 1% increase with a 36 mg/dl increase in mean plasma glucose concentrations.

There are more than 30 different methods for determination of GHbs. These methods separate hemoglobin from GHb using technique based on charge differences (ion-exchange chromatography, HPLC, electrophoresis, IEF), structural differences (affinity chromatography and immunoassay), or chemical analysis (photometry and spectrophotometry). The result in all is expressed as percentage of total Hb.

Ion exchange chromatography separates Hb variants on the basis of charge. The cation exchange resin (negatively charged) packed in disposable minicolumn has an affinity for Hb, which is positively charged. The patient’s sample is hemolyzed and an aliquot of the hemolysate is applied to the column. A buffer is applied and the eluent collected. Here GHb is less positively charged than other so will elute first than other. The eluted GHb (A1a, 1b and 1c, collectively A1) are measured in spectrophotometer. Other Hbs are also measured after subsequent elution and the HbA1 is expressed as percentage of total.

HPLC can be used for separation and quantitation of HbA1c and other fractions. HPLC employs, cation exchange chromatography.

Agar gel electrophoresis on whole blood hemolysates at pH 6.3 provides good resolution of HbA and HbA₁. The gel contains negatively charged moieties that interacts with the hemoglobin. After 25 to 35 minutes, the GHb separates on the cathodic side of HbA. Quantification is done by scanning densitometry at 415 nm.

The hemoglobin variant separate on IEF on the basis of their migration in gel containing pH gradient on acrylamide gel slabs.

Immunoassay with the principle of immunoinhibition are used like ELISA where antibodies are raised and used to inhibit other fraction in one hand and capture and detection antibodies are used to determine HbA1c.

Affinity gel columns are used to separate GHb, which binds to the column, from the nonglycated fraction. M-Aminophenylboronic acid is immobilized by cross linking to beaded agarose or another matrix (e.g., glass fiber). The boronic acid reacts the cis-diol groups of glucose bound to Hb to form a reversible five member ring complex thus selectively holding the GHb on the column. The nonglycated Hb does not bind. Sorbitol is then added to elute the GHb. Absorbance of the bound and nonbound fractions measured at 415 nm is used to calculate the percentage of GHb. Nonglycated Hb does not bind and is removed In a wash step. The sorbitol competes for boronate binding sites. 

(Source: Tietz Clinical Chemistry, 4th Edition)

For borate affinity assay, packed blood cells are mixed with hemolysate reagent that contain borate buffer. Glycated Hb is assayed from this hemolysate.