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HbA1c

HbA1c. 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. Hemoglobin. Human adult hemoglobin

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HbA1c

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  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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  21. 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)

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

  23. 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

  24. 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

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

  26. 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

  27. 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

  28. 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

  29. 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

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

  31. 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

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

  33. 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

  34. 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

  35. 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

  36. 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

  37. 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

  38. 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

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

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