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Glycated hemoglobin (HbA1c)

Glycated hemoglobin (HbA1c). Introduction. Diabetes mellitus, as previously stated is a condition of hyperglycemia. It is estimated that this condition affects 2.5-5% of the population and is considered to be the fifth leading cause of death in the U.S.

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Glycated hemoglobin (HbA1c)

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  1. Glycated hemoglobin (HbA1c)

  2. Introduction • Diabetes mellitus, as previously stated is a condition of hyperglycemia. • It is estimated that this condition affects 2.5-5% of the population and is considered to be the fifth leading cause of death in the U.S. • The disease is associated with a number of serious micro and macro-vascular complications involving the eyes, kidneys, heart and blood vessels, and may greatly impair the quality of life or shorten the life-span of the person afflicted.

  3. Introduction • There is a relationship between control of the glucose concentration fluctuation and the progression of the disease complications. • There should be a method to quantify accurately and objectively the degree of altered blood glucose control over a long period of time.

  4. Glycosylated hemoglobin • In adults, hemoglobin is a mixture of three forms: Hb A1, Hb A2,Hb F, with Hb A1 predominating. • Hemoglobin A1 consists of three subforms: Hb A1a, Hb A1b and Hb A1c, with Hb A1c predominating. • The term glycated hemoglobin describes a chemically stable conjugate of any of the forms of hemoglobin with glucose. • Glycated forms of hemoglobin are formed slowly, nonenzymatically and irreversibly at a rate that is proportional to the concentration of glucose in the blood.

  5. Glycation: Nonenzymatic addition of a sugar residue to amino groups

  6. Significance of test • By testing for glycosylated hemoglobin, the doctor discovers what the average blood glucose level has been for the previous 2 to 3 months. • This is especially valuable when monitoring diabetics whose blood sugars change dramatically from day-to-day and to monitor long-term diabetic control. • HbA1c are a better and less expensive measure of long term glucose control than repeated fasting glucose test. • Unaffected by exercise or recent food ingestion

  7. Glycosylated hemoglobin • Blood levels of Glycated hemoglobin • Depends • on the life span of red cells • the blood glucose concentration

  8. Specimen • EDTA is the anticoagulant of choice for all methods. • No special preparation, fasting specimens are not required. • Most methods require cell lysis with a hemolyzing reagent provided by the manufacturer prior to loading. • Typically, whole blood may be stored up to 7 days at 2-8 C. • heparinized samples • should be assayed within 2 days and may not be suitable for other methods (electrophoresis • NOTE: Blood glucose level reflects the previous few hours glycemic state , glycated albumin reflects 10-14 days glycemic state, While HbA1c reflects the longest 2-3 months glycemic state.

  9. Methods: 1. Gel electrophoresis. 2. Immunoassay. 3. Affinity chromatography. 4. Cation exchange chromatography.

  10. Cation exchange chromatography • Hemoglobin variants are separated based on charge difference • Bed • cation exchange resin (negatively charged)

  11. Principle of the method • After preparing the hemolysate , Where the labile fraction is eliminated, hemoglobins are retained by a cation exchange resin. Hemoglobin A1c is specifically eluted after washing away the hemoglobin A1a+b fraction, and is quantified by direct photometric reading at 415 nm . The estimation of the relative concentration of HbA1c is made by the measure of the total hemoglobin concentration by direct photometric reading at 415nm .

  12. Procedure:shake thoroughly and let it stand at room temperature for 10-15 minutes. Column Preparation: Remove the upper cap of the column and then snap the tip off the bottom.Using the flat end of a pipette, push the upper disc down to the resin surface taking care not to compress it. Let the column drain completely to waste. • Hemolysate preparation :

  13. Separation and reading of HbA1c: • 1. Carefully pipette on the upper filter: 2. In order to drain any sample residue left above the upper disc pipette: 3. Pipette: 4. Place the column over a test tube and add: Shake thoroughly and read the Abs of HbA1c at 415nm against distilled water . The absorbance is stable for at least one houre.

  14. Reading of Hb total: • Calculation: %HbA1c = Shake thoroughly and read the Abs at 415nm against distilled water . The absorbance is stable for at least one houre. A of HbA1c fraction * V HbA1c %100 A of Hb TOTAL * V Hb total

  15. Reference range • Glycated Hb is expressed as a percentage.

  16. Interpretation of Glycated hemoglobin • Glycated Hb should be routinely monitored at least every 3 month in all insulin treated patients • Causes of elevated HbA1c: • Uncontrolled DM • Increase in HbF • Increase in Triglycerides • Lead toxicity • Renal failure+ Hemodialysis • Causes of decreased HbA1c: • Causes of decrease RBC life span (hemolytic or bleeding • Hemodilution e.g.: pregnancy.

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