Glucose & Glycated Hb (Hba1c)

1. Glucose & GlycatedHb (Hba1c) Lab 4 M. Zaharna Clin. Chem. Lab

2. Introduction • All body tissues can utilize glucose, the principle and almost exclusive carbohydrate circulating in blood. • The glucose level in the blood is maintained within a narrow range under diverse conditions by regulatory hormones • Measurement of glucose is one of the most commonly performed procedure in most hospital chemistry laboratories • The most frequently encountered disorder of carbohydrates metabolism is a high blood glucose due to DM M. Zaharna Clin. Chem. Lab

3. Disorders In Carbohydrate Metabolism • The various disorders in carbohydrate metabolism may be grouped into several categories dependent primarily upon laboratory findings: • those associated with a raised plasma glucose concentration (hyperglycemia); • Those associated with decreased plasma glucose concentration (hypoglycemia); M. Zaharna Clin. Chem. Lab

4. Estimates of The Glucose Concentration • Estimates of the glucose concentration in blood are required to: • help in the diagnosis of diabetes mellitus • management of DM patients • and monitoring of treatment in DM patients • It is also essential for the detection and proper management of hypoglycemia, a condition encountered much less frequently • neonatal hypoglycemia • or any other condition in which there is abnormal carbohydrate metabolism in the body. M. Zaharna Clin. Chem. Lab

5. Methods Of Analysis • Until 15 or 20 years ago, the majority of the quantitative tests for glucose determination depended upon the oxidation of glucose by hot, alkaline copper solutions or solutions of potassium ferricyanide. • These were replaced by the ortho-toluidine test and later by enzyme methods employing either glucose oxidase or hexokinase. M. Zaharna Clin. Chem. Lab

6. Methods Of Analysis • Enzymatic methods give a “true” glucose determination because of the high specificity of an enzyme for a particular substrate. • There should be fewer interfering substances when measuring the glucose concentration in serum using enzyme methods. M. Zaharna Clin. Chem. Lab

7. Methods Of Glucose Analysis • Method 1: Benedict’s; qualitative, semiquantitative • This procedure, sensitive to total reducing compounds present in urine, yields red Cu2O and yellow CuOH precipitates. • Method 2: o-Toluidine; quantitative • The o-toluidine reaction is based on the ability of many aromatic amines in acid solutions to condense with the aldehyde group of glucose to form glycosamines. • Increased absorbance at 630 nm M. Zaharna Clin. Chem. Lab

8. Enzymatic Methods • Method 3: Hexokinase (HK); quantitative • Involves two coupled reactions and two enzymes. • Yield 1 mol of NADH or NADPH for each mole of glucose that is oxidized. (Read Absorbance at 340nm) • Method 4: Glucose oxidase coupled reaction, Quantitative • One of the most frequently used specific glucose methods uses two coupled enzyme reactions • Method 5: Glucose dehydrogenase; quantitative, • Increased absorbance at 340 nm is related to glucose concentration (NADH produced) M. Zaharna Clin. Chem. Lab

9. Specimen Collection • Regardless which method of glucose determination is employed, precautions in the sample collection are required to prevent the utilization of glucose by cells. • The glucose loss, upon standing in a warm room, may be as high as 10 mg/dL per hour. • The decrease in serum glucose concentration is negligible if the blood sample is kept cool and the serum separated from the clot within 0.5 hours of drawing. M. Zaharna Clin. Chem. Lab

10. Specimen Collection • The addition of 2 mg sodium fluoride per mL blood to be collected will prevent glycolysis for 24 hours. M. Zaharna Clin. Chem. Lab

11. Specimen Collection And Preparation • Serum, plasma is suitable for samples. • Whole blood and hemolysis are not recommended for use as a sample. • Freshly drawn serum is the preferred specimen. • Stability: Serum heparin or EDTA-plasma (with addition of glycolytic inhibitor) : • 2 days at 20–25oC • 7 days at 2–8oC M. Zaharna Clin. Chem. Lab

12. Glycated Hemoglobin (HbA1C) M. Zaharna Clin. Chem. Lab

13. 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. M. Zaharna Clin. Chem. Lab

14. 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. M. Zaharna Clin. Chem. Lab

15. Glycosylated Hemoglobin • In adults, hemoglobin is a mixture of three forms: Hb A1, Hb A2, and 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. M. Zaharna Clin. Chem. Lab

16. Glycosylated Hemoglobin • As blood glucose levels rise, the increase in glycosylation of proteins is proportional to both the level of glucose and the lifespan of the protein being affected. • With consideration of RBC's lifespan, glycosylated hemoglobin has been accepted as a measurement which reflects, better than FBS determinations, • the mean daily blood glucose concentration • and the degree of carbohydrate imbalance over the preceding period (90-120 days) . M. Zaharna Clin. Chem. Lab

17. Glycosylated Hemoglobin • Today, hemoglobin A1C (HbA1c) testing is performed to monitor diabetics suspected of having irregular control over their glucose level. • Glycated hemoglobin measurements are, however, influenced by conditions that affect the life span of the hemoglobin molecule, such as sickle cell disease and hemolytic disease, which can falsely decrease glycated hemoglobin results. M. Zaharna Clin. Chem. Lab

18. 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. • Whereas a fasting blood sugar may be influenced by the patient’s recent adherence to the prescribed treatment regimen, M. Zaharna Clin. Chem. Lab

19. The glycosylated hemoglobin is irreversible; it shows what type of diabetic control has occurred over several months. • If the diabetes is controlled (basically an HbA1C lower than 7%), the test should be done every 3-6 months. M. Zaharna Clin. Chem. Lab

20. M. Zaharna Clin. Chem. Lab

21. 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. M. Zaharna Clin. Chem. Lab

22. Normal Values of HbA1C • Nondiabetic adult: 3-5 % • Diabetic adult: <7% M. Zaharna Clin. Chem. Lab