A presentation to physicians on using Hba1c in clinical practice.

1. Glycosylated hemoglobin
HbA1c
Mathew John MD, DM, DNB
Endocrinologist
Providence Endocrine & Diabetes
Specialty Centre

2. Some terms
• A1c : Glycated hemoglobin = glycosylated
hemoglobin= glycohemoglobin(US)
• IFCC: International Federation of Clinical Chemistry
• NGSP: National Glycohemoglobin Standardisation
Programme
• DCCT : Diabetes Control and Complications Trial
• ADAG : A1c derived average glucose

3. Hemoglobin
HbA0(α2 ß2)
HbA2(α2δ2) HbF(α2γ2)
90 %
HbA1
HbA1c
Non ezymatically glycosylated form of human
hemoglobin, taking place under physiological
conditions, at a specific site on the protein

4. Terminology
• Hb: hemoglobin
• HbA1: is a series of glycated variants resulting from
attachment of various carbohydrates to N terminal
valine of Hb
• Glycation results in increased negative charge and hence
runs fast on electrophoresis systems
Pickup & Williams , Textbook of Diabetes

5. GHb: glycated hemoglobin
1. HbA1a1: fructose 1,6 diphosphate N terminal valine
2. HbA1a2: glucose 6 phosphate N terminal valine
3. HbA1b: unknown carbohydrate N terminal valine
4. HbA1c: (60-80%): attachment of glucose to N
terminal amino acid valine of the beta chain of
hemoglobin
Total glycated Hb: HbA1c+ sugar Non N terminal sites

6. Amadori rearrangement of glucose
molecule.

7. Relationship of HbA1C to Risk of
Microvascular Complications
Diabetes Control and Complications Trial
(DCCT)
15
13 Retinopathy
Relative Risk (%)
Nephropathy
11
Neuropathy
9 Microalbuminuria
7
5
3
1
6 7 8 9 10 11 12
HbA1C (%)
Skyler JS. Endocrinol Metab Clin North Am. 1996;25:243-254.

8. Factors affecting HbA1c
Falsely elevated values
• HbF or HbG
• Uremia ( BUN > 85 mg/dl)
• Hypertriglyceridemia( cation exchange +, EP-)
• Alcohol
• High bilirubin( cation exchange+, HPLC+)
• Aspirin
• Splenectomy, Aplastic anemia

9. Factors affecting HbA1c
Falsely low HbA1c
• HbC, HbS
• Hemolytic anemias
• Pregnancy
• Acute/ Chronic blood loss
• Vitamin E/C
• Dapsone
• Severe nephropathy ( shorten RBC survival)
Glycated hemoglobin monitoring BMJ 2006 ; 333;586-8

10. Age specific targets
Age HbA1c target
<6 years 7.5 –8.5 %
6-12 years < 8%
13-19 years <7.5 %
Silverstein J, Diabetes Care; 28;2005

11. Current HbA1c recommendations
Normal IDF ADA AACE
A1c* <6% <6.5% <7% <6.5%
Preprandial <100 <110 90-130 <110
Postprandial <140 <155 <180 <140

12. What does HbA1c represent ?

13. Patients with variable diurnal profiles
can have the same A1c
Roger Mazze DIABETES TECHNOLOGY & THERAPEUTICS
Volume 10, Supplement 1, 2008

14. Relationship between FPG, PPG
and HbA1c
80
Postprandial Fasting Hyperglycemia
Contribution (%)
60
40
20
0
1 2 3 4 5
(<7.3) (7.3-8.4) (8.5-9.2) (9.3-10.2) (>10.2)
Monnier L, Diabetes Care 2003;26
HbA1c quintiles

15. ADAG study
A1c Derived Average Glucose
• Define the mathematical relationship between A1c and
average glucose levels
• 507 subjects : 268 with type 1 diabetes, 159 with type 2
diabetes and 80 non diabetic subjects
• A1c at end of 3 months compared with average glucose
during the previous 3 months
• From 2 day CGMS 4 times+7 point SMBG 3 times/week
Nathan D Diabetes Care 31:1-6, 2008

16. ADAG study
• Approx 2700 values/subject in 3 months
• Linear regression analysis between A1c and AG
values provided the tightest correlations
AG (mg/dl) = 28.7X A1C-46.7 ( R2 0.84, P 0.0001)

17. ADAG study
Estimated average glucose ( e AG)
mg/dl mmol/L DCCT
135
170
205
240
275
310
Nathan D Diabetes Care 31:1-6, 2008

18. Hba1c represents more recent
sugars

19. Mean blood sugars vs. ADAG
ADAG
MBG
ADAG : A1c Derived Average Glucose

20. Methods of measuring HbA1c
• Ion exchange chromatography : low pressure
HPLC
• Electrophoretic methods
• Immunoturbimetric methods
• Affinity methods
• Chemical methods: e.g thiobarbituric method
• Electrospray iontophoresis
• Mass spectroscopy
• Reversed phase HPLC

21. Can we use HbA1c for diagnosis
of diabetes ?

22. Cut offs
Fasting plasma glucose cut offs for definition of IGT and DM
Normal IGT Type 2 diabetes
100 mg/dl 126 mg/dl

23. Diagnosis of diabetes
• Diagnosis of diabetes has always been glucose centric
: based on FBS, 2 hr post glucose , RBS
• National Diabetes Data Group (NDDG) 1979 : relied on
distributions of glucose levels
• Based on their association with decompensation
to “overt” or symptomatic diabetes
FPG > 140 mg/dl
PPG > 200 mg/dl

24. 1997
1997, the Expert Committee on the Diagnosis and
Classification of Diabetes Mellitus refocused attention on
the relationship between glucose levels and the presence
of long-term complications as the basis for diagnosis of
diabetes
The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Report of the Expert
Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 1997;20:1183–
1197

25. Expert Committee on the Diagnosis and
Classification of Diabetes Mellitus
Committee recommended that the FPG cut point be
lowered to 126 mg/dl (7.0 mmol/l) so that this cut
point would
• Represent a degree of hyperglycemia that was “similar”
to the 2HPG value and diagnosis with either measure
would result in a similar prevalence of diabetes in the
population
• Introduced the concept of IFG and IGT
The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Report of the Expert
Committee on the Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 1997;20:1183–
1197

26. Pathophysiologic cut offs
Looked at 3 studies which compared glycemia to risk of
retinopathy
• Egyptian population (n 1,018)
• Pima Indians (n 960),
• U.S. National Health and Nutrition Examination Survey
(NHANES) population (n 2,821)

27. FPG/PPG /HbA1c vs. Retinopathy
U.S. National Health and Nutrition Examination Survey
(NHANES) population (n 2,821)

28. Current use of HbA1c
• Monitor long term glycemic control
• Adjust therapy
• Assess the quality of diabetes care
• Predict the risk for the development of complications

29. HbA1c for diagnosis of diabetes
• HbA1c correlates with retinopathy
• There was a stronger correlation between A1C and
retinopathy than between fasting glucose levels and
retinopathy
• Similar correlation between A1c and Retinopathy has
been seen in DCCT/ UKPDS trials
• 1997 Expert Committee recommended against using
A1C values for diagnosis in part because of the lack of
assay standardization

30. 2009 :International Expert Committee Report on
the Role of the A1C Assay in the Diagnosis
of Diabetes
DIABETES CARE, VOLUME 32, NUMBER 7, JULY 2009
Recommends that HbA1c be adopted as one of the
diagnostic criteria for diabetes

31. What has happened between
2003 and 2009 ?
Advances in instrumentation and standardization,
the accuracy and precision of A1C assays at least
match those of glucose assays
International Expert Committee Report on the Role of the A1C Assay in the Diagnosis of Diabetes. DIABETES CARE,
VOLUME 32, NUMBER 7, JULY 2009

32. New reference standards
• International Federation of Clinical Chemistry (IFCC)
• Measures “ pure” A1c
• Pure A1c: N-[1deoxylfructos-1-yl]) hemoglobin beta
chain, abbreviated as DOF hemoglobin
• Expressed as mmol/mol of Hb
• HbA1c of 5% would now be about 33 mmol/mol, and an
8% A1C would be about 58 mmol/mol.

33. Pitfalls with glucose measurement
• The measurement of glucose itself is less accurate
and precise than most clinicians realize
• 41% of instruments have a significant bias from the
reference method that would result in potential
misclassification of 12% of patients
• Potential preanalytic errors owing to sample handling
• Lability of glucose in the collection tube at room
temperature
International Expert Committee Report on the Role of the A1C Assay in the Diagnosis
of Diabetes. DIABETES CARE, VOLUME 32, NUMBER 7, JULY 2009

34. Advantages of HbA1c
• HbA1c is stable after collection
• New reference method to calibrate all A1C assay
instruments should further improve A1C assay
standardization in most of the world between- and
within-subject
• Coefficients of variation have been shown to be
substantially lower for A1C than for glucose
measurements
• The variability of A1C values is also considerably less
than that of FPG levels, with day-to-day within-person
variance of 2% for A1C but 12–15% for FPG
International Expert Committee Report on the Role of the A1C Assay in the Diagnosis
of Diabetes. DIABETES CARE, VOLUME 32, NUMBER 7, JULY 2009

35. Advantages of HbA1c
• Convenience for the patient and ease of sample
collection for A1C testing
• Relatively unaffected by acute (e.g., stress or illness
related) perturbations in glucose levels
International Expert Committee Report on the Role of the A1C Assay in the Diagnosis
of Diabetes. DIABETES CARE, VOLUME 32, NUMBER 7, JULY 2009

36. Cut off of HbA1c for diagnosis of
diabetes
• Cut offs at which the prevalence of retinopathy increases
• NHANES data and DETECT 2 study

37. DETECT 2 study
Prevalence of retinopathy by 0.5% intervals and severity of retinopathy in participants aged 20–79 years. NPDR, nonproliferative
diabetic retinopathy. Adapted with permission from (S.C., personal communication).
19,000 subjects from nine countries
The glycemic level at which the prevalence of “any” retinopathy begins to rise above
background levels and for the more diabetes-specific “moderate” retinopathy, was
6.5% when the data were examined in 0.5% increments

38. Cut off of HbA1c
• A1C level of 6.5% is sufficiently sensitive and specific
to identify individuals who are at risk for developing
retinopathy and who should be diagnosed as diabetic
• A1C level is at least as predictive as the current FPG and
2HPG values.

39. Should we use of HbA1c to
diagnose diabetes in our set up ?

40. Limitations
• Cost may preclude routine use
FBS + PPBS: Rs. 60/ -
HbA1c : Rs. 275/ -
• Standardized methods and instrumentation
POC instruments
• Hemoglobin variants
• Any condition that changes red cell turnover, such as
hemolytic anemia, chronic malaria, major blood loss, or
blood transfusions
• A1C levels appear to increase with age

41. Limitations
• Discordance with standard diagnostic criteria
• The prevalence of diabetes in some populations
may not be the same when diagnosis is based on
A1C compared with diagnosis with glucose
measurements, and one method may identify different
individuals than the other.
• Ethnic variations in HbA1c at same glucose levels exists

42. “ Prediabetes”
• Once A1c is used to diagnose diabetes, “ prediabetes” or
IGT/ IFG may be obsolete
• HbA1c between 6 and 6. 5 % :
higher risk for developing diabetes
more effective interventions

43. Practical considerations
• POC instruments are not to be used to make this
diagnosis
• Always confirm using the same tests
• Intermethod variability is reported to still be a potential
source of inaccuracy

44. Point of care instruments
• DCA Vantage
• Nycocard
• In2it (BioRad)
• A1cNow( Bayer)

45. Methods for HbA1c
The better and best
Electrospray iontophoresis
Mass spectrometry
HPLC
CV : 2-3 %
Immunoassay
methods
CV 5-6 %
•Point of care ( POC)
Instruments
• Colorimetry

46. BioRad D10
• A1C quantitation in the presence of HbS,
HbC and HbF
• Optimized to minimize interference from
carbamylation, lipemia and labile A1C
• Traceable to the IFCC reference method
• NGSP Certified

47. Words of wisdom
• HbA1c and mean glucose corroborate abnormal glucose
metabolism, but it requires self monitoring ( or CGMS) to
detect the location and magnitude of the abnormalities
• HbA1c and SMBG should be considered together, with
each complementing the information provided by the
other
Peacock I J Clin Path 1984

48. HbA1c for all patients ?
Added Glimiperide 1 month
2 mg/day
HbA1c
Started
On Metformin Glargine 14
1000 mg/day units/day
3 months

49. HbA1c for all patients ?
Glargine dose
18 units/day
HbA1c
Glargine 14 units/day
Metformin
1000 mg/day
Glimiperide 2 mg/day

50. 2010 Consensus Statement on the Worldwide
Standardization of the HbA1C Measurement
• HbA1c test results should be standardized worldwide
• The IFCC reference system for HbA1c represents the
only valid anchor to implement standardization
• HbA1c results are to be reported by clinical laboratories
worldwide in SI units (mmol/mol, no decimals) and
derived NGSP units (%, one decimal)
DIABETES CARE, VOLUME 33, NUMBER 8, AUGUST 2010

51. High Commission Labs Pvt Ltd,
69, Park Road, NY
Name: Kuttapan J, 45 yrs Male
HbA1c: 8.0% ( Biorad D10 variant 11)
eAG2: 183 mg/dl
IFCC HbA1c3 : 58mmol/mol
1.Biorad D10 is a DCCT aligned method
2.e AG are derived from ADAG study by Nathan et al. Nathan D Diabetes Care 31:1-6, 2008
3.IFCC A1c is estimated from a regression equation . From Jeppsson J-O, Clin Chem Lab Med
2002;40:78-8

52. Thank you