GLYCATED HEMOGLOBIN (A1c/HbA1c/DOF- Hb )

1. GLYCATED HEMOGLOBIN(A1c/HbA1c/DOF-Hb) Methodologies, clinical reporting & analytical issues Dr SikanaarHayat Khan

2. Learning outcomes To improve upon the clinical usage of HbA1c What are the different methodologies for HbA1c? What should be out the analytical goals & standardization practices? What interpretation issues we can face in labs? How result should be reported & what treatment goals we should highlight as “chemical pathologists”? Dr Sikandar Hayat Khan

3. Formation Hbglycation occurs when glucose attaches to one or both of the N-terminal valines of the b-chain to form a SCHIFF’S BASE The process is fast and unstable and proceeds to form anIRREVERSIBLE KETOAMINE through Amadori rearrangement After a period of weeks, ADVANCED GLYCATION ENDPRODUCTS are formed and broken down Dr Sikandar Hayat Khan

4. CLINICAL USE Dr SikandarHayat Khan

5. Points to ponder???? Can HbA1c be used for screening diabetes mellitus in all individuals? Can methodologies affect clinical decision making? What cut-offs are recommended for diagnosing diabetes mellitus? What cut-offs be recommending for monitoring diabetes mellitus? Dr Sikandar Hayat Khan

6. Clinical use • Diagnosis of diabetes mellitus • Monitoring of diabetes therapy • SPECIAL SITUATIONS • PREGNANCY • ICU Dr Sikandar Hayat Khan

7. Why glycated hemoglobin??? Dr Sikandar Hayat Khan

8. Diagnosing diabetes International Expert Committee Report, drawn from the International Diabetes Federation (IDF) European Association for the Study of diabetes (EASD) American Diabetes Association (ADA), suggests the A1C level OF 48 mmol/mol (6.5 DCCT %) AS A DIAGNOSTIC LEVEL (The Committee Report further states that, when A1C testing cannot be done, the fasting and glucose tolerance tests be done) Dr Sikandar Hayat Khan

9. Diagnosing diabetes Dr Sikandar Hayat Khan

10. Diagnosing diabetes Dr Sikandar Hayat Khan

11. Points to ponder ??? What is the cost comparison of fasting blood glucose to glycated hemoglobin? Can POCT devices for A1c be used for diagnosis? Which parameter has more inter-lab variation? Which parameter has more biological variation? Dr Sikandar Hayat Khan

12. Points to ponder??? Dr SikandarHayat Khan

13. Monitoring diabetes STABLE DIABETES UNSTABLE DIABETES Individuals with unstable glucose & A1C treatment goals, like: Change of treatments Not meeting glycemic goals Individuals with stable glucose & A1C treatment goals TWICE A YEAR QUARTERLY Dr SikandarHayat Khan

14. Monitoring diabetes therapy Personalized Dr Sikandar Hayat Khan

15. SPECIAL SITUATIONS Hughes et al. Diabetes Care. 2014 Nov;37(11):2953-9. doi: 10.2337/dc14-1312. DIAGNOSIS OF DIABETES DURING PREGNANCY continues to require fasting and glucose tolerance measurements for gestational diabetes, and not the glycated hemoglobin Dr SikandarHayat Khan

16. Hemoglobin types • Correct answer: Reliance of different parameter like glucose/fructosamine? • In these patients a shortened red blood cell lifespan is typically encountered, which leads to a falsely low hemoglobin A1c • How diabetic monitoring be carried out in subjects known to have hemoglobinopathies with no/minimal amount of HbA? • Different cut-offs? • Different methodologies? • Reliance of different parameter like glucose/fructosamine? Dr Sikandar Hayat Khan

17. Methodologies Dr SikandarHayat Khan

18. Methods for glycated hemoglobin Dr Sikandar Hayat Khan

19. Methods for glycated hemoglobin CHARGE BASED STRUCTURE BASED Boronate affinity chromatography Affinity electrophoresis Immunoassays • Cation-exchange resin chromatography • Electrophoresis • Cat ion-exchange HPLC Dr SikandarHayat Khan

20. Methods for glycated hemoglobin CHARGE BASED STRUCTURE BASED Boronate affinity chromatography Less susceptible to interference from Hb variants Immunoassay Antibodies target the B-N terminal of first 4-10 AA of the glycated hemoglobin Less susceptible to interference from Hb variants Caution with regards to certain ethnicities • More susceptible to interference from Hb variants • More susceptible to post-translational modifications (e.g., carbamylation and acetylation) Dr SikanadarHayat Khan

21. POCT devices Nycocard i-chroma DCA Vantage In2 It (Biorad) A1cNow (Bayer) Dr SikandarHayat Khan

22. POCT devices Dr Sikandar Hayat Khan

23. ANALYTICAL GOALS & STANDARDIZATION Dr SikandarHayat Khan

24. Points to ponder??? Do we face inter-lab variation in our A1c reporting? What specific reason do we have for inter-lab variation? Do we think of calibration issues while installing a new kit program in our labs? Does it matter? What IFCC has to say about calibration? What is primary reference method for HbA1c ? Dr Sikandar Hayat Khan

25. An assay standardization issue Hanas R et al. Diabetes Care. 2002;25:2110-2111. Multiple changes in treatment and associated psychological changes were faced by patients due to A1c methodology& calibration changes Dr Sikandar Hayat Khan

26. Precision targets Program must be able to achieve a CV below 3% at HbA1c levels of 6% and 9% Manufacturers’ assays should be able to achieve a CV below 5% Reference: IFCC AND AACC International Standardization Committee Dr Sikandar Hayat Khan

27. Comparison of precision Check your kits and methods? Everybody is different? Dr Sikandar Hayat Khan

28. A1c Inter-lab variation Mosca A at al. Global standardization of glycated hemoglobin measurement: the position of the IFCC Working Group. Clin Chem Lab Med.2007;45(8):1077-80. CVs of 5%–7% has been shown for HbA1c values between 6% and 10% Poor between-lab agreement can also be found for the same manufacturer’s method IFCC established a Working Group (IFCC WG-HbA1c)to achieve international standardization of HbA1c measurement Dr Sikandar Hayat Khan

29. IFCC standardization program (HbA1c) Achievements of this WG includes: • Development of highly purified primary Reference Materials(HbA1c and HbA0) by Institute for Reference Materials and Measurements (IRMM) • A reference measurement procedure for HbA1c has been developed as: - Proteolytic digestion of red cell hemoglobinsFOLLOWED BY -Quantitative peptide mapping by: • HPLC-mass spectrometry OR • HPLC-capillary electrophoresis It has been voted on by the National Societies affiliated to the IFCC and published as an ‘‘APPROVED IFCC REFERENCE MEASUREMENT PROCEDURE’’ Dr Sikandar Hayat Khan

30. IFCC standardization program (HbA1c) Achievements of this WG includes: • Development of highly purified primary Reference Materials (HbA1c and HbA0) by Institute for Reference Materials and Measurements (IRMM) • A reference measurement procedure for HbA1c has been developed as: - Proteolytic digestion of red cell hemoglobinsFOLLOWED BY -Quantitative peptide mapping by: • HPLC-mass spectrometry OR • HPLC-capillary electrophoresis It has been voted on by the National Societies affiliated to the IFCC and published as an ‘‘APPROVED IFCC REFERENCE MEASUREMENT PROCEDURE’’ Dr Sikandar Hayat Khan

31. IFCC standardization program (HbA1c) c) Provision to IFCC lab network for further development of secondary reference materials • Two experiments are performed every year in which materials are distributed to the laboratories for: • Comparison purposes • To assign HbA1c values to candidate calibrators and controls • The network has developed: • SET OF RULES for the certification of reference values • To obviate uncertainties in CALCULATION OF CALIBRATORS Dr DikandarHayat Khan

32. IFCC standardization program (HbA1c) d) Comparison studies have been performed between the IFCC reference measurement laboratories and the existing Designated Reference Method (DCMs) • These studies found stable relationships between the IFCC and different (DCM) systems and the corresponding regression equations (the ‘‘master equations’’) were published Dr DikandarHayat Khan

33. IFCC standardization program (HbA1c) e) Secondary Reference Materials (SRMs) have been produced in the form of panels of fresh and frozen whole blood and distributed to the manufacturers and to laboratories performing DCMs to anchor their methods to the IFCC reference system Dr DikandarHayat Khan

34. IFCC standardization program (HbA1c) IRMM IFCC lab netwrok IFCC reference measurement system and traceability chain for HbA1c Dr DikandarHayat Khan

35. Name and units for the IFCC standardized HbA1c test Recently, a recommendation by the IFCC-IUPAC Committee on Nomenclature, Properties and Units (C-NPU) has been prepared that relates to the: • SYSTEMATIC • UNITS FOR HbA1c(as measured by IFCC ref procedure) Dr Sikandar Hayat Khan

36. Name and units for the IFCC standardized HbA1c test (NOMENCLATURE) What compound do we have in mind once we say glycated hemoglobin? Reference: IFCC-IUPAC (C-NPU) document on glycated hemoglobin The IFCC WG-HbA1c agrees with the use of this nomenclature because it gives tracebility to the glycated hemoglobin to original IFCC reference procedure Fraction of the b-chains of hemoglobin that has a stable hexose adduct on the N-terminal amino acid valine Thus it can be expressed as ‘‘Hemoglobin beta chain(Blood)—N-(1-deoxyfructos-1-yl) ’’ i.e., bN1-deoxyfructosyl-hemoglobin (DOF-Hb) Dr Sikandar Hayat Khan

37. Name and units for the IFCC standardized HbA1c test(UNITS) • IFCC supports the use of ‘‘millimole per mole’’ will be chosen instead of ‘‘percent’’ (%) • According to IFCC to avoid confusion ? • Converting to SI units • Broad range for explanation of results Dr Sikandar Hayat Khan

38. RESULT REPORTING & treatment goals Dr SikandarHayat Khan

39. Reporting HbA1c as eAG *(Goldstein et al, ClinChem 1986;32:B64) **(Nathan et al, NEJM 1984;310;341) DCCT trial and UKPDS data indicated a direct relationship between HbA1c and Mean Blood Glucose 1% change of HbA1c = 25 -35% change in average plasma glucose* MBG = 33.3 (HbA1c) - 86 (r=0.958)** Major advantage by reporting HbA1c as an eAG is that both physicians and patients understand glucose Dr DikandarHayat Khan

40. Translating HbA1c as eAG *Diabetes Care 2008; 31:`473-1478 **ClinChem Lab Me 2008’46:1617-1623 The A1c Assay into estimated average glucose values* A1c determination in the A1c Derived Average Glucose (ADAG)** Dr DikandarHayat Khan

41. eAG limitations high glycator will have a much higher A1c than the low glycator and vice versa * *Hempe JM et al. J Diabetes Complications 2002;16(5):313-20. • Ethnic data (Most studies on Caucasians) • No data from • Children • Pregnancy • Renal failure • Concept of “glycation status” High glycatorsvs low glycators Dr DikandarHayat Khan

42. Measuring eAG eAG (mmol/l) = 1.59 × A1C − 2.59 Dr DikandarHayat Khan

43. Measuring eAG • eAG Endorsements • American Diabetes Association (ADA) • International Diabetes Federation (IDF) • American Association of Clinical Chemistry (AACC) • International Federation of Clinical Chemistry (IFCC) • European Association for the study of Diabetes (EASD) Dr Sikandar Hayat Khan

44. IFCC vs DCCT confusion Dr Sikandar Hayat Khan

45. IFCC vs DCCT confusion *Guidelines by ADA, EASD, IFCC and IDF • Switch to IFCC units • HbA1c is to be reported in the IFCC units* • IFCC reporting was introduced in Europe except for the UK in 2003 • UK carried out dual reporting from 1st June 2009 until 1 October 2011 (Still going on in most set ups) Dr Sikandar Hayat Khan

46. IFCC vs DCCT confusion So if u have a DCCT certified kit reporting a HbA1c result of 9% (75 mmol/mol) then what will be his IFCC A1c values = ???? (6.85%) 74.86 mmol/mol The 2010 ADA Standards of Medical Care in Diabetes added the A1c ≥ 48 mmol/mol (≥6.5 DCCT %) as another criterion for the diagnosis of diabetes Dr DikandarHayat Khan

47. A reporting pattern for HbA1c Dr DikandarHayat Khan

48. Reporting data for A1C • The data to be provided with HbA1c must include following: • Results as per DCCT (%) • Results as per IFCC (mmol/mol) • Methodology type • HbA1c types included • eAG • Appropriate references • Diagnostic & therapeutic cut-offs • Interpretation & opinion Dr Sikandar Hayat Khan

49. Diagnostic & therapeutic goals Dr DikandarHayat Khan

50. Interpretation issues Dr SikandarHayat Khan