1. HbA1c

2. Glycated proteins Monitoring long term glucose control Retrospective index of the integrated plasma glucose Is not subject to the wide fluctuations Adjunct to blood glucose determination not for the diagnosis of diabetes mellitus

3. Hemoglobin Human adult hemoglobin –HbA 97%of the total, HbA2 2.5%, HbF 0.5% Minor hemoglobins –HbA1a HbA1b, HbA1c collectively referred to as HbA1 –fast hemoglobins

4. Glycation –Nonenzymatic addition of a sugar residue to amino groups –Neoglycoprotein, Glycation HbA1a1; –fructose 1, 6 diphosphate HbA1a2 – glucose 6 phosphate HbA1b –pyruvic acid HbA1C –glucose –major fraction 80% of HbA1

6. Hb A0 –Glycation at lysine residues, or α chain –measured by affinity chromatography Blood levels of Glycated hemoglobin –Depends on the life span of red cells the blood glucose concentration

7. Glycated Hb Free of day to day fluctuations Unaffected by exercise or recent food ingestion Recent glucose values provide larger contributions to glycated Hb than earlier values. The plasma glucose in the preceding one month makes up 50% of the HbA1c whereas days 60- 120 determine only 25%. blood glucose over the preceding 6-8 week

8. Interpretation of Glycated hemoglobin sources of errors –Low Glycated hemoglobin hemolytic disease shortened red blood cell survival recent blood loss High Glycated hemoglobin –Iron deficiency anemia –the effect of hemoglobin variants such as Hb F, S and C

9. Carbamylated Hb Labile intermediates pre Hb A1C, Schiff base depends on the specific method of analysis Labile fraction –changes rapidly with acute changes in blood glucose –spuriously alter Glycated Hb values

10. Pre-Hb A1c –amounts to 5-8% of total Hb A1 in normal people –8-30 % in patients with diabetes Glycated Hb should be routinely monitored at least every 3 month in all insulin treated patients

11. Clinical utility of Glycated Hb For glycemic control to decrease long term complications of diabetes mellitus To reduce the risk of retinopathy, nephropathies, and neuropathy to delay the onset and to slow the progression of these complications

12. Study; a 10% lower Hb A1c was assocated with a 45% lower risk of retinopathy An index of long term blood glucose concentration in patients with diabetes mellitus The goal is blood glucose control

13. Methods for the determination of glycated hemoglobin selection of method –Including sample volume, patient population, and cost most widely used technique –affinity chromatography In the United States methods based on charge

15. Total glycated hemoglobin (A1+A0), HbA1 (HbA1a1+A1a2+A1b+A1c). In Europe –HPLC and ion-exchange with less use of affinity chromatography

16. Ion exchange chromatography Hemoglobin variants are separated based on charge difference Bed –cation exchange resin (negatively charged) Procedure –hemolysis of the patient sample, a buffer is applied and the eluent collected. –Elution The ionic strength and pH of the eluent buffer are selected so that glycated hemoglobins are less positively charged

17. Ion exchange chromatography A second buffer of different ionic strength to elute the more positively charged main Hb fraction –this is read as total Hb glycated Hb is expressed as percentage of total Hb

18. Ion exchange chromatography Modifications –Flow rates are accelerated by centrifugation –Batch technique agitation of resin with hemolysate to adsorb Hb A –Using two different buffers to separate HbA1a+b from A1c

19. Factors affecting Ion exchange chromatography The temperature of the reagents and columns –thermostatting the columns –applying a correction factor Control of pH and ionic strength Sample storage condition different minicolumns exhibit wide variability in performance

20. Factors affecting Ion exchange chromatography The labile pre-Hb A1 fraction –produce elevated results HbF –elutes with HbA1 produce falsely elevated results Alteration of charge on Hb –carbamylated Hb, alcoholism,lead poisoning and acetylated Hb HbS,HbC and their glycated derivatives; misleading low values for HbA1

21. HPLC The principle –Cation exchange chromatography Procedure –Application of hemolysate –Elution stepped elution –phosphate buffer of increasing ionic strength –Detection absorbance at 415 and 690 nm

22. HPLC Good resolution of Hb A1 a+b from HbA1c –with sodium phosphate-cyanide buffer at different pH values Elution as one peak (HbA1) –A rapid system –evaluation with a dual wavelength detector at 405 for HbA1 and 546 for HbA –Interference Hb variants Hb C Hb F carbamylated and acetylated forms of Hb

23. HPLC Quantification –Integrating the area under the peaks An automated system –Step gradients using three phosphate buffers of increasing ionic strength –Detection at 415 and 690nm –both Hb A1c and HbA1 is reported –Variant Hb are resolved (Hb F, S and C)

24. HPLC HPLC methods –have excellent precision –recommended as reference method interference –Carbamylated and acetylated Hb and possibly other derivatives slightly higher results

25. Electrophoresis Agar gel at pH 6.3 resolution of Hb A and HbA1 The gel contains negatively charged moieties Quantification performed by scanning densitometry at 415 nm HbA1c is also commercially available Results agree with that of HPLC or column but are less precise Minor variations in pH, ionic strength or temperature have little effect on results HbF migrates the same as HbA1and causes falsely elevated value Hb C and S do not The labile form should be removed

26. Isoelectric focusing Principle; migration in gel containing a pH gradient Matrix; acrylamide gel pH range of 6-8 On completion the gels are fixed and then scanned by a microdensitometer Hb A1c resolved from HbA1a, A1b, S and F Results showed close agreement with other methods The equipment is expensive

27. Immunoassay Anti serum raised against purified human HbA1c Available methods RIA format Enzyme immunoassay format Agglutination inhibition

28. Immunoassay Antibodies raised against the Amadori product of glucose (ketoamine linkage) plus the first few amino acids at the N- terminal of β-chain Agglutinator; a synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c, light scattering

29. Immunoassay Excellent precision The antibodies do not recognize labile intermediates or other glycated hemoglobins Other Hb variants such as HbF, A2, S, carbamylated Hb are not detected. Correlate well with HPLC but exhibit lower values Due to different calibration, detection by HPLC of substances other than HbA1c

30. Affinity chromatography Principle –m-aminophenyl boronic acid is immobilized by cross linking to beaded agarose or other matrix (e.g., glass fiber) –The boronic acid react with the cis-diol groups of glucose –Dissociation By Sorbitol –Detection Absorbance of bound and non bound fractions measured at 415 nm

32. Affinity chromatography Advantage –No interference non glycated Hb –Negligible interference from the labile intermediate form –Unaffected by variations in temperature –Reasonably good precision –Hemoglobin variants Hb F, S, and C produce little effect

33. Affinity chromatography Report –Affinity methods measure total glycated Hb –Commercially available systems are calibrated to also report a HbA1c standardized value

34. Specimen –Patients need not be fasting –Venous blood containing EDTA, oxalate, or fluoride –Whole blood my be stored at 4°C for up to 1 week – storage at -20°C or 80°C is not recommended

35. heparinized samples –should be assayed within 2 days and may not be suitable for other methods (electrophoresis)

36. Preparation of hemolysate –Packed cell Centrifuge –remove the plasma and buffy coat Wash with saline –Removal of labile glycated Hb Incubation of RBC in saline in buffer solutions at pH 5 to 6 by dialysis or ultrafiltration of hemolysate

37. Preparation of column –Bring the column to room temperature –Remove the caps –Pour off upper buffer –Add equilibration buffer let drain and discard the eluate

38. Assay standardization The absence of a reference method and a single glycated Hb standard has generated confusion Interlaboratory comparisons are not possible calibration –significantly improves precision and facilitates direct comparison of results obtained by different methods

39. Assay standardization Calibrator –lyophilized hemolysate assayed by a precise HPLC method for Hb A1c adoption of a universal standard will enhance the clinical utility of glycated Hb

40. Reference interval Values for glycated Hb are expressed as a percentage of total Hb Three major glyacted Hb species –HbA1, HbA1c, or total glycated Hb Reference intervals vary depending on –method –the glycated Hb component –whether the labile fraction is included

42. Reference intervals Reference intervals show some increase with age poorly controlled diabetes –values may extent to twice the upper limit of the reference interval Values grater than 20% should prompt further studies There is no specific level of HbA1c below which the risk of diabetic complications is eliminated completely

43. Reference intervals Each laboratory should establish its own nondiabetic reference interval Assay precision is important; each 1% change = 25-35 mg/dl change