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HbA1c : glycosylated hemoglobin
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
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
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