GLYCATED HEMOGLOBIN(HbA1C)

Prepared by:
BASALINGAPPA.B.G.
2ND M.Sc. Medical Biochemistry
JSS Medical college Mysore
 TITLE OF HbA1C
• Terminology, Definition and Description.
• Principle
• Methods
• Structure
• Sample Preparations And Maintenance
• Advantages and disadvantages
• Normal and abnormal values
• Clinical Significance
Terminology
• Hb: haemoglobin
• HbA1: is a series of glycated variants resulting from
attachment of various carbohydrates to N terminal
valine of Hb.
Glycosylation: enzymatic addition of any sugar
(glucose)to a protein molecule.
• Glycation: when once glucose attached ,it is not removed
from the hemoglobin,therefore it remains inside the
erythrocyte, so the life span of RBCS(120Days )non enzymatic
Some terms
• A1c : Glycated haemoglobin together called HbA1
fraction.
• IFCC: International Federation of Clinical Chemistry
• NGSP: National Glycohaemoglobin Standardisation
Programme
• DCCT : Diabetes Control and Complications Trial
• ADAG : A1c derived average glucose
 Definition.
• Glycated haemoglobin is a form of hemoglobin that
is measured primarily to identify the three month
average plasma glucose concentration.
• The test is limited to a three month average because
the lifespan of a red blood cell is three
months(120Days).
• It is formed in a non-enzymatic glycation pathway by
haemoglobin's exposure to plasma glucose.
Structure of hemoglobin
 Principle
Glycation of proteins is a frequent occurrence, but in the case of
haemoglobin, a non enzymatic reaction occurs between glucose and the N-end
of the beta chain.
This forms a Schiff base which is itself converted to 1-deoxyfructose.
• A Schiff's base a nitrogen analog of an aldehyde or ketone in which the
C=0.group is replaced by C=N-R (amine) group. It is Usually formed by
condensation of an aldehyde or ketone with a primary amine.

• A1c is a weighted average of blood glucose levels during the life of the red
blood cells (120 days). Therefore glucose levels on days nearer to the test
contribute substantially more to the level of A1c than the levels in days
further from the test.

• This is also supported by data from practice showing that HbA1c levels
improved significantly by 20 days from the start of the intensification of
glucose-lowering treatment
 Where R, may be an alkyl or an aryl group. Schiff bases that contain aryl
substituents are substantially more stable and more readily synthesized,
while those which contain alkyl substituents are relatively unstable. Schiff
bases of aliphatic aldehydes are relatively unstable and readily
polymerizable1,2 while those of aromatic aldehydes having effective
conjugation are more stable

The formation of a Schiff base from an aldehydes or ketones is a

reversible reaction and generally takes place under acid or base catalysis,
or upon heating.
 What does the A1c test measure ?
• Normally, some of the glucose (sugar) in the bloodstream attaches
itself to proteins in our body..

• Once the sugar is attached to the haemoglobin, it stays there for the
life of the red blood cell, which is about 120 days (3 months).

• The higher the level of blood sugar, the more sugar attaches to
haemoglobin and the higher the per cent of haemoglobin which is
glycosylated (HbA1c).

• This is why the results are given as a percentage (for example. 7.5 %).
• When blood glucose levels are high, glucose molecules attach to the
haemoglobin in red blood cells. The longer hyperglycaemia occurs
in blood, the more glucose binds to haemoglobin in the red blood
cells and the higher the glycated haemoglobin.

• Once a haemoglobin molecule is glycated, it remains that way. A

build-up of glycated haemoglobin within the red cell, therefore,
reflects the average level of glucose to which the cell has been
exposed during its life-cycle.

• Measuring glycated haemoglobin assesses the effectiveness of

therapy by monitoring long-term serum glucose regulation.
Alternative names:
• Beta N‐(1‐deoxyfructos‐1‐yl)
haemoglobin .
• haemoglobin A1c, HbA1c, glycated
haemoglobin,
• glycohaemoglobin,
• Glycosylated haemoglobin.
 Different Hbs
1. Fetal Hemoglobin – HbF
2. Adult Hemoglobin – HbA
3. Sickle cell disease – HbS
4. Hemoglobinopathies – HbC, HbE
5. Glucose in the blood reacts with the
Hemoglobin A to form Glycated Hb.
 Why is this test performed?
• The HbA1c test is a way to assess blood
glucose control over time.
• It represents an “average” blood glucose level
over the previous 100 days.
• However, it does not replace the need for
heme blood glucose monitoring on a daily
basis
 What does HbA1c measure?

• HbA1c measures how much glucose is stuck to the

haemoglobin in your blood.
• This tells you what your average blood glucose level has been
for the past 2 - 3 months.
• The HbA1c result is given as a percentage figure. By
monitoring your HbA1c levels regularly you can see if your
diabetes control is improving or deteriorating.
• It is always important to know your most recent HbA1c level:
– 1. If your result is higher than normal, the chances of developing diabetic
complications are increased.
– 2. If your result is lower than normal, the chances of developing diabetic
complications are decreased.
 Reference values of HbA1c
1. 4.5 -5.6%- Normal
2. 5.7-6.4%- prediabetics
3. More than 6.5%- Diabetics
4. 6.6-7%- Adequate control
5. 7-8%- Inadequate control
6. More than 9% - Very poor control.
7. According to ADA(American Diabetic
Association criteria's)
 Methods
• A number of techniques are used to measure haemoglobin
A1c.
• Laboratories use:
• (HPLC): The HbA1c result is calculated as a ratio to total
haemoglobin by using a chromatogram.
• Immunoassay
• Enzymatic
• Capillary electrophoresis
• Boronate affinity chromatography
 TYPES OF HPLC DEPENDS ON:
Molecular weight of
solute

Water solubility of
solute

Polarity of solute

Ionic and non-ionic

character of solute
Practical considerations

• POC instruments are not to be used to make this diagnosis

• Always confirm using the same tests

• Inter method variability is reported to still be a potential

source of inaccuracy
Sample preparation
• Whole blood primer:
• Reconstitute With 1mL of DI Water
• Allow to stand for 10-15 minutes; swirl gently
to dissolve .
• Stable for 1day at 2-8Celcius
 HbA1C Calibrators:
• 2 Calibrators ( Level 1& Level 2)
• Reconstitute Each Vial with 7mL of cold
calibrator Diluent.
• Allow to stand for 5-10minutes; Swirl gently to
dissolve.
• Stable for 7 days at 2-8C
Lypochek controls:
• Reconstitute Each vial with 0.5 mL of DI Water
• Allow to stand for 5-10 minutes; swirl gently
to dissolve.
• Stable for 7 days at 2-8C
• Dilute 1:300Prior to Analysis
• (5mL of control in 1.5 ml of wash /diluent
solution).
 Liquichek Controls:
• After opening Vial ,Stable for 14 days At 2-8C
• Dilute 1:200 prior to Analysis
Whole Blood Samples
• Samples Should be Collected in Vaccume
Collection tubes Containing EDTA.
• Stable For 7days At 2-8C Or 3 days at room
Temperature (15-30C)
• Allow Sample Tubes At Room temperature(15-
30C).No Sample preparation required.
Daily Maintenance Of HbA1C Pre Run
• Check that the correct method(HbA1C) is
installed.
• Check buffer/wash levels, lot numbers, And
line positions.
• Check reagent on-board expiration dates.
• Check cartridge injection Count And lot
Number.
• Check for leaks during pressure check
• Check external waste tank level.
 Continued,,,,,,,,,,
• Check pump pressure with pump running
(maintain screen):
• Flow rate at 1.5mL/min,50% buffer 2.
• Pump pressure should not fluctuate more
than 5%.
• Prime the lines if needed.
• Check printer paper is there or not to supply.
Point of care instruments
• DCA Vantage
• Nycocard
• In2it (Bio-Rad)
• A1cNow( Bayer)
Bio-Rad D10
1.A1C quantitation in the presence of HbS,
HbC and HbF.
2. Optimized to minimize interference from
carbamylation, lipemia and labile A1C.
3. Traceable to the IFCC reference method.
4. NGSP(National glycohaemoglobin
standardisation programme) Certified.
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
 Description of HbA1C.
Structural and chemical investigations elucidated that glucose, in the open chain
format, binds to the N‐terminal to form an aldimine (Schiff base) before
undergoing an Amadori rearrangement to form a more stable ketamine.

• It is an organic reaction ,describing the acid or base catalysed

isomerization or rearrangement reaction of N-glycoside of an aldose or
glucosamine i.e.to the corresponding 1 amino –deoxy ketoses.
• Normal adult haemoglobin consists predominantly of
• HbA1 (α2β2),
• HbA2 (α2δ2)
• HbF (α2γ2),
• About 6% of total HbA is termed.
HbA1, which in turn is made up of
HbA1a1,
HbA1a2,
HbA1b
HbA1c.
• These fractions are defined by their electrophoretic and chromatographic
properties, which differ slightly from those of the major component HbA0,
despite the amino acid sequences of HbA1 and HbA0 being identical.
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 like) valine of the beta chain of
Haemoglobin.
Total glycated Hb: HbA1c+ sugar Non N terminal sites
 Advantages of HbA1c
• Index of long-term control over 120
days and not a snap shot like PG
• Can be done at any time of day
• Not influenced by diet, exercise,
emotional disturbances on test day
• Useful index in clinical trials
• Useful if missed drugs / default diet
• Useful in stress hyperglycemia
 Continued,,,,,,,,,,,
• For HbA1c ,Fasting sample is not required.
• Low intra individual variability <2%.
• HbA1C sample is also stable; While blood sugar
level is lowered unless precautions are taken.
• HbA1C value is not altered by Acute factors, while
many factors will affect blood sugar values.
• HbA1C is a better index for predicting
complications. Because of all these
reasons,HbA1C has become the preferred test
nowadays.
 Limitations of HbA1c
• Cannot be an emergency room test to
titrate Insulin or OHA dosage
• Cannot register hypoglycemia
• More sensitive to sin than repentance –
if it is elevated it confirms poor control,
if it is boarder line, it cannot assure
good control in the recent past.
• Not sensitive enough for use in GDM
•  Anemia, Uremia, Pregnancy
Factors affecting HbA1c
• Acute hyperglycemia
• Severe aneamia
• Gestational diabetes
• Life span of the RBC
• Abnormal Hb like S-Hb, Hb C
• Serum opalescence -↑TG
• On the method of estimation
Blood Glucose Monitoring
Type Frequency Sample

Type 2 DM Monthly FPG / PPG

Type 1 DM 4-6 times 6th hourly

initially to 4th hourly
Stabilized Twice a week. 3 samples

Pregnancy Once a week. FPG / PPG

Peri-operative 4-6 times 6th hourly

a day to 4th hourly
Correlation of MPG - HbA1c
Mean Plasma Glucose = (33.3 x HbA1C%) - 86

HbA1C % Mean BG mg %

5 80.5
7 147.1
9 213.7
11 280.3
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 DE compensation
to “overt” or symptomatic diabetes
FPG > 140 mg/dl
PPG > 200 mg/dl
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
How can I make use of this information?

Keep a record of your HbA1c and note down each one.

By recording them, you will be able to monitor your

progress

Keeping good control of your blood sugars helps reduce

the risk of long - term complications of diabetes

improves your general health and well being.

ANY QUESTIONS..??