1. GLYCATED HEMOGLOBIN (A1c/HbA1c/DOF-Hb)
Methodologies, clinical reporting & analytical issues
7/27/2019
Dr Sikanaar Hayat 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”?
7/27/2019
Dr Sikandar Hayat Khan

3. Formation
Hb glycation 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 an IRREVERSIBLE KETOAMINE through Amadori rearrangement
After a period of weeks, ADVANCED GLYCATION ENDPRODUCTS are formed and broken down
7/27/2019
Dr Sikandar Hayat Khan

4. CLINICAL USE
Dr Sikandar Hayat Khan
7/27/2019

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?
7/27/2019
Dr Sikandar Hayat Khan

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

7. Why glycated hemoglobin???
7/27/2019
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)
7/27/2019
Dr Sikandar Hayat Khan

9. Diagnosing diabetes
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Dr Sikandar Hayat Khan

10. Diagnosing diabetes
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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?
7/27/2019
Dr Sikandar Hayat Khan

12. Points to ponder???
Dr Sikandar Hayat Khan

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

14. Monitoring diabetes therapy
Personalized
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Dr Sikandar Hayat Khan

15. SPECIAL SITUATIONS
DIAGNOSIS OF DIABETES DURING PREGNANCY continues to require fasting and glucose tolerance measurements for gestational diabetes, and not the glycated hemoglobin
Hughes et al. Diabetes Care Nov;37(11): doi: /dc
Dr Sikandar Hayat Khan

16. Hemoglobin types
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?
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
7/27/2019
Dr Sikandar Hayat Khan

17. Methodologies
Dr Sikandar Hayat Khan
7/27/2019

18. Methods for glycated hemoglobin
CHARGE BASED
CATION-EXCHANGE RESIN CHROMATOGRAPHY
ELECTROPHORESIS
CATION-EXCHANGE HPLC
STRUCTURE BASED
BORONATE AFFINITY CHROMATOGRAPHY
AFFINITY ELECTROPHORESIS
IMMUNOASSAYS
CHEMICAL METHODS
REFERENCE METHOD
IFCC TANDEM MASS SPECTROMETRIC ASSAY
POCT
IMMUNOASSAY
7/27/2019
Dr Sikandar Hayat Khan

19. Methods for glycated hemoglobin
CHARGE BASED
STRUCTURE BASED
Cation-exchange resin chromatography
Electrophoresis
Cat ion-exchange HPLC
Boronate affinity chromatography
Affinity electrophoresis
Immunoassays
7/27/2019
Dr Sikandar Hayat Khan

20. Methods for glycated hemoglobin
CHARGE BASED
STRUCTURE BASED
More susceptible to interference from Hb variants
More susceptible to post-translational modifications (e.g., carbamylation and acetylation)
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
Caution with regards to certain ethnicities
7/27/2019
Dr Sikanadar Hayat Khan

21. POCT devices Nycocard i-chroma DCA Vantage In2 It (Biorad)
A1cNow (Bayer)
7/27/2019
Dr Sikandar Hayat Khan

22. POCT devices
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Dr Sikandar Hayat Khan

23. ANALYTICAL GOALS & STANDARDIZATION
Dr Sikandar Hayat Khan
7/27/2019

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 ?
7/27/2019
Dr Sikandar Hayat Khan

25. An assay standardization issue
When the test was brought in-house and results reported began to be slightly higher than they had been, a trend appeared in which the overall results drifted down (ie, patients intensified their efforts).
The opposite effect was observed when the calibration was changed and results appeared lower than they had been previously. Then a trend appeared in which the overall results drifted up (ie, patients allowed their glycemic control to deteriorate).
Clearly patients (and physicians) respond to changes in hemoglobin A1c results.
Multiple changes in treatment and associated psychological changes were faced by patients due to A1c methodology& calibration changes
Hanas R et al. Diabetes Care. 2002;25:
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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?
7/27/2019
Dr Sikandar Hayat Khan

28. A1c Inter-lab variation
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
Mosca A at al. Global standardization of glycated hemoglobin measurement: the position of the IFCC Working Group. Clin Chem Lab Med.2007;45(8):
7/27/2019
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 hemoglobins FOLLOWED 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’’
7/27/2019
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 hemoglobins FOLLOWED 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’’
7/27/2019
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 Dikandar Hayat 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 Dikandar Hayat 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 Dikandar Hayat Khan

34. IFCC standardization program (HbA1c)
IRMM
IFCC lab netwrok
IFCC reference measurement system and traceability chain for HbA1c
Dr Dikandar Hayat 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)
7/27/2019
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?
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)
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
7/27/2019
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
7/27/2019
Dr Sikandar Hayat Khan

38. RESULT REPORTING & treatment goals
Dr Sikandar Hayat Khan
7/27/2019

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

40. Translating HbA1c as eAG
The A1c Assay into estimated average glucose values*
A1c determination in the A1c Derived Average Glucose (ADAG)**
*Diabetes Care 2008; 31:`
**Clin Chem Lab Me 2008’46:
Dr Dikandar Hayat Khan

41. eAG limitations
Ethnic data (Most studies on Caucasians)
No data from
Children
Pregnancy
Renal failure
Concept of “glycation status”
High glycators vs low glycators
MBG
Mean HbA1c
95% CI HbA1c
154 mg/dl
7.0% %
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):
Dr Dikandar Hayat Khan

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

43. eAG (estimated average glucose)
Measuring eAG
HbA1c
eAG (estimated average glucose)
(%)
(mmol/mol)
(mmol/L)
(mg/dL) 5 31
5.4 (4.2–6.7)
97 (76–120) 6 42
7.0 (5.5–8.5)
126 (100–152) 7 53
8.6 (6.8–10.3)
154 (123–185) 8 64
10.2 (8.1–12.1)
183 (147–217) 9 75
11.8 (9.4–13.9)
212 (170–249) 10 86
13.4 (10.7–15.7)
240 (193–282) 11 97
14.9 (12.0–17.5)
269 (217–314) 12
108
16.5 (13.3–19.3)
298 (240–347) 13
119
18.1 (15–21)
326 (260–380) 14
130
19.7 (16–23)
355 (290–410) 15
140
21.3 (17–25)
384 (310–440)
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)
7/27/2019
Dr Sikandar Hayat Khan

44. IFCC vs DCCT confusion
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Dr Sikandar Hayat Khan

45. IFCC vs DCCT confusion 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)
*Guidelines by ADA, EASD, IFCC and IDF
7/27/2019
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 Dikandar Hayat Khan

47. A reporting pattern for HbA1c
Dr Dikandar Hayat 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
7/27/2019
Dr Sikandar Hayat Khan

49. Diagnostic & therapeutic goals
Dr Dikandar Hayat Khan

50. Interpretation issues
Dr Sikandar Hayat Khan
7/27/2019

51. Consideration Your method type? Clinical need? Patient type?
Charge based?
Non-charge based?
POCT ?
Clinical need?
Diagnosis?
Monitoring?
Patient type?
Type-1 diabetic
Type-2 diabetic
Pregnancy?
Underlying ailments?
Red cell disorder (Hemoglobinopathies?)
Uremia?
IDA?
7/27/2019
Dr Sikandar Hayat Khan

52. Concept of interpretation
A1c
RBC related
Glucose bonding
Analytical targets
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Dr Sikandar Hayat Khan

53. Patient condition affecting A1c results
Increase
Decrease
Splenectomy *#
Thalassemia *#
HbF*#
IDA*
Uremia (Carbamylated Hb)*
Alcoholism*
Hyperbilirubinemia*
Hypertriglyceridemia*
Lead poisoning*
Large dose of salicylates (Acetylated Hb)*#
Opiate addiction*
Hemolytic anemia
Acute blood loss
HbC*
HbS*
Pregnancy
*ion-exchange
#electrophoretic method
7/27/2019
Dr Dikandar Hayat Khan

54. Medications affecting A1c results
Glycation
Sulfation
Acetylation
Carbamylation
7/27/2019
Dr Sikandar Hayat Khan

55. Influences on “Glycation Process”
RBC’s half-life
Hemolysis
Hemoglobinopathies
Anemia
B12/Folate deficiency
Blood glucose
Glucose permeability (Door to glucose entry into cell) Hb G
Permeability of the RBC to glucose influences the amount of glycation and could explain the discordance noted in some hematologically normal people with diabetes
7/27/2019
Dr Sikandar Hayat Khan

56. Assessment Question Your Reply
Any method of A1c reporting can be used for diagnosis of diabetes mellitus (YES/NO)
DOF-Hb has been endorsed by IFCC for use IFCC (YES/NO)
What A1c cut-off has been recommended by DCCT for diabetes diagnosis?
SI units for reporting A1c are __________
Glycation rates are not different among various ehnicities (TRUE/FALSE)
7/27/2019
Dr Sikandar Hayat Khan

57. Assessment Question Your Reply
A1c reporting needs to be standardized to improve accuracy (YES/NO)
Drugs causing hemolysis can decrease Aic results (YES/NO)
Precision recommended by IFCC as a n anlytical target for A1c should be?
A patient with long standing iron deficiency anemia can have lower A1c results? (TRUE/FALSE)
What kind of bias the IFCC result will have in comparison to DCCT-certified results?
7/27/2019
Dr Sikandar Hayat Khan

58. Assessment Question Your Reply
Hypertriglyceridemia and hyperbilirubinemia can increase A1c results? (YES/NO)
IFCC reference method for A1c is ______
Boronate affinity chromatography is a charge based method. (TRUE/FALSE)
Stable diabetes is monitored by twice yearly testing for A1c (TRUE/FALSE)
Precision issues in case of A1c are more pronounced at higher values (TRUE/FALSE)
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Dr Sikandar Hayat Khan

59. Thanks a lot
7/27/2019
Dr Sikandar Hayat Khan