Glycosylated hemoglobin

HbA1c

Mathew John MD, DM, DNB

Endocrinologist

Providence Endocrine & Diabetes Specialty Centre

Some terms

• A1c : Glycated hemoglobin = glycosylatedhemoglobin= glycohemoglobin(US)

• IFCC: International Federation of Clinical Chemistry• IFCC: International Federation of Clinical Chemistry

• NGSP: National Glycohemoglobin Standardisation Programme

• DCCT : Diabetes Control and Complications Trial

• ADAG : A1c derived average glucose

Hemoglobin

HbA0(α2 ß2)90 %

HbA2(α2δ2) HbF(α2γ2)

Non ezymatically glycosylated form of human hemoglobin, taking place under physiological conditions, at a specific site on the protein

HbA1

HbA1c

Terminology

• Hb: hemoglobin

• HbA1: is a series of glycated variants resulting from attachment of various carbohydrates to N terminal valine of Hbvaline of Hb

• Glycation results in increased negative charge and hence runs fast on electrophoresis systems

Pickup & Williams , Textbook of Diabetes

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 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

Amadori rearrangement of glucose molecule.

Relationship of HbA1C to Risk of Microvascular Complications

Rela

tive R

isk (

%)

Retinopathy

Nephropathy

Neuropathy

15

13

11

Diabetes Control and Complications Trial(DCCT)

Skyler JS. Endocrinol Metab Clin North Am. 1996;25:243-254.

Rela

tive R

isk (

%)

Neuropathy

Microalbuminuria

HbA1C (%)

11

9

7

5

3

16 7 8 9 10 11 12

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

Factors affecting HbA1c

Falsely low HbA1c

• HbC, HbS

• Hemolytic anemias

• Pregnancy

• Acute/ Chronic blood loss• Acute/ Chronic blood loss

• Vitamin E/C

• Dapsone

• Severe nephropathy ( shorten RBC survival)

Glycated hemoglobin monitoring BMJ 2006 ; 333;586-8

Age specific targets

Age HbA1c target

<6 years 7.5 –8.5 %

6-12 years < 8%

Silverstein J, Diabetes Care; 28;2005

6-12 years < 8%

13-19 years <7.5 %

Current HbA1c recommendations

Normal IDF ADA AACE

A1c* <6% <6.5% <7% <6.5%

Preprandial <100 <110 90-130 <110Preprandial <100 <110 90-130 <110

Postprandial <140 <155 <180 <140

What does HbA1c represent ?

Patients with variable diurnal profiles can have the same A1c

Roger Mazze DIABETES TECHNOLOGY & THERAPEUTICS

Volume 10, Supplement 1, 2008

Relationship between FPG, PPG and HbA1c

Contrib

ution (%

)

60

80

Postprandial Fasting Hyperglycemia

HbA1c quintiles

Contrib

ution (%

)

(<7.3) (7.3-8.4) (8.5-9.2) (9.3-10.2) (>10.2)

0

20

40

1 2 3 4 5

Monnier L, Diabetes Care 2003;26

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 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

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)AG (mg/dl) = 28.7X A1C-46.7 ( R2 0.84, P 0.0001)

ADAG study

mg/dl mmol/L

Estimated average glucose ( e AG)

DCCT

Nathan D Diabetes Care 31:1-6, 2008

135

170

205

240

275

310

Hba1c represents more recent sugars

Mean blood sugars vs. ADAG

ADAG

ADAG : A1c Derived Average Glucose

ADAG

MBG

Methods of measuring HbA1c

• Ion exchange chromatography : low pressure

HPLC

• Electrophoretic methods

• Immunoturbimetric methods

• Affinity methods• Affinity methods

• Chemical methods: e.g thiobarbituric method

• Electrospray iontophoresis

• Mass spectroscopy

• Reversed phase HPLC

Can we use HbA1c for diagnosis of diabetes ?

Cut offsFasting plasma glucose cut offs for definition of IGT and DM

Normal IGT Type 2 diabetes

100 mg/dl 126 mg/dl

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 levelsdistributions of glucose levels

• Based on their association with decompensation

to “overt” or symptomatic diabetes

FPG > 140 mg/dl

PPG > 200 mg/dl

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 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

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 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

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• U.S. National Health and Nutrition Examination Survey

(NHANES) population (n 2,821)

FPG/PPG /HbA1c vs. Retinopathy

U.S. National Health and Nutrition Examination Survey

(NHANES) population (n 2,821)

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

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 • 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

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

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

New reference standards

• International Federation of Clinical Chemistry (IFCC)

• Measures “ pure” A1c

• Pure A1c: N-[1deoxylfructos-1-yl]) hemoglobin betachain, abbreviated as DOF hemoglobin

• Expressed as mmol/mol of Hb• 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.

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 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

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-subjectwithin-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

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

Cut off of HbA1c for diagnosis of diabetes

• Cut offs at which the prevalence of retinopathy increases

• NHANES data and DETECT 2 study

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

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• A1C level is at least as predictive as the current FPG and

2HPG values.

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

Limitations

• Cost may preclude routine use

FBS + PPBS: Rs. 60/ -

HbA1c : Rs. 275/ -

• Standardized methods and instrumentation

POC instruments 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

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 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

“ 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 higher risk for developing diabetes

more effective interventions

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

Point of care instruments

• DCA Vantage

• Nycocard

• In2it (BioRad)

• A1cNow( Bayer)

Methods for HbA1cThe better and best

HPLC

Electrospray iontophoresisMass spectrometry

•Point of care ( POC) Instruments • Colorimetry

Immunoassaymethods CV 5-6 %

HPLCCV : 2-3 %

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

• HbA1c and mean glucose corroborate abnormal glucose metabolism, but it requires self monitoring ( or CGMS) to detect the location and magnitude of the abnormalities

Words of wisdom

• HbA1c and SMBG should be considered together, with each complementing the information provided by the other

Peacock I J Clin Path 1984

HbA1c for all patients ?

On Metformin1000 mg/day

Added Glimiperide 2 mg/day

Started Glargine 14 units/day

1 month

HbA1c

1000 mg/day units/day

3 months

HbA1c for all patients ?

Glargine dose18 units/day

HbA1c Glargine 14 units/day Metformin1000 mg/dayGlimiperide 2 mg/day

HbA1c

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

Name: Kuttapan J, 45 yrs Male

HbA1c: 8.0% ( Biorad D10 variant 11)

High Commission Labs Pvt Ltd,High Commission Labs Pvt Ltd,High Commission Labs Pvt Ltd,High Commission Labs Pvt Ltd,69, Park Road, NY

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

Thank you