Background study is to mainly focus on the use,


Recent studies from different experts have helped
clarify the role and use of glycated haemoglobin in the management of diabetes. Diabetes,
being one of the rapidly arisen diseases over the world can have massive consequences
in the future as according to WHO organisation, diabetes has affected over 400 million
people worldwide and the numbers are predicted to nearly double to it and
become the seventh leading
cause of death in 2030 (WHO, 2016).  

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mellitus (DM) is a metabolic disorder with heterogenous causes, which is
characterised by chronic hyperglycaemia due to defects in insulin action,
secretion or the combination of them both. This abnormality of insulin results
in affecting the metabolism of carbohydrates, protein and fat (World Health
Organisation, 2011). There are two main types of diabetes; type 1 diabetes,
which is depended on insulin and type 2 diabetes which is not dependant on
insulin (Ahmed, 2011). Type 1 diabetes is an autoimmune disorder, which is a
consequence of an absolute insulin deficiency as a result of pancreatic beta
cell destruction by T-cell mediation (Kawasaki, 2014). Type 2 on the other hand is caused by insulin
resistance. The pancreas produces insulin but it either is not enough or the insulin
is not used by the body effectively (Isley and Molitch, 2005). Overall, this
indicates that the body will build up glucose as there won’t be enough of
insulin to process glucose or the body becomes resistance to the effect of
insulin. This build up can lead to an increased risk of microvascular and
macrovascular diseases or other serious health complications such as, blindness,
kidney failure and lower limbs amputation etc. Therefore, it is crucial to use
highly standardised tests such, as HbA1c as this test can provide strong
indication of both diabetes and pre-diabetes in a timeframe, where actions can
be made immediately (Niflioglu et al., 2012)

The aim of this study is to mainly focus on the use, power
and efficacy of the glycated haemoglobin (HbA1c) in the diagnosis of diabetes

Diagnostic criteria for DM:

mentioned above that hyperglycaemia is a sign of diabetes, it will end up showing
a few major symptoms like diabetic retinopathy and diabetic keto acidosis in
type 1 diabetes. Diabetes patients can also experience polyuria, polydipsia,
fatigues and weight loss (Ahmed, 2011).

For many
years, the main option carried out for the diagnosis of diabetes have been the
measurement of the glycaemic control level that individual exhibits, which
indicates the presence of glucose in their blood (American Diabetes Association,
2013). Tests included in this option are fasting plasma glucose (FPG) test and
Oral glucose tolerance test (OGTT). HbA1c was later introduced and approved by
WHO in 2011. The results of these obtained from WHO can be seen in table 1.

The OGTT was
considered as a “gold standard” test, which made it possible to assess patients’
ability to metabolise glucose (Sacks, 2011). OGTT is a two hours plasma glucose
indication. It requires patients to fast overnight in order to measure their
venous plasma glucose concentration. A load of 75g anhydrous glucose is then
consumed by patients and a glucose concentration after 2 hours is re-measured (WHO,
2016). There are two categories where patients may fall into; IGT or IFG.  These individuals are described as having prediabetes
and are at significant risk of developing DM (mostly Type 2) and the
complications associated with it.

Table 1:
WHO recommendations for the diagnostic criteria for diabetes or pre-diabetes
(WHO, 2016).














* Values
of venous plasma glucose after 2 hours of ingestion of 75 g oral glucose load

limitation with OGTT and FPG tests is that even though blood sugar levels can
vary throughout the day and over time, these tests only measure blood glucose at
one point at time. This indicate that a good measure of average plasma sugar
over a particular time is not provided (Sacks, 2011). Therefore, the use of glycated haemoglobin HbA1c was introduced, which
gives more accurate diabetes diagnosis. HbA1c is not only helpful for the diagnosis
of diabetes but it also manages the glycaemic control without any special
preparation such as fasting. Thus, it has been deemed as a new “gold standard” diagnostic
tool for diabetes mellitus because of its properties (Alqahtani et al., 2013).

In 2005,
when WHO revealed their criteria, it included a statement that HbA1c should not
be adopted as a diagnostic test as the challenges of measurement accuracy
outweighed the convenience of its use. However later in 2011, they reviewed
their previous decision to recommend HbA1c as diagnostic marker for diabetes
and they came up with a conclusion that HbA1c can be used as a diagnostic test
for diabetes. A
value of 6.5% (48mmol/L) is recommended as the cut-off point for diagnosing
diabetes. A value less than 6.5% or to be more precise, a value between 5.7%
and 6.4% indicates pre-diabetes. If the value undercomes between 6.6.5, there
is a high risk of developing diabetes. More than 7% is considered to be high
thus risky.

t is important to consider that HbA1c values below 6.5% and 5.7%
do not reliably exclude the presence of diabetes and prediabetes,



Structure and process of the Glycated

HbA1c is
synthesised by a non- enzymatic reaction of glucose molecule binding with
N-terminal valine of the haemoglobin beta chains. This binding forms an
aldimine (Schiff base or labile HbA1c) which itself
is converted to 1-deoxyfructose. Schiff base then undergoes an Amadori rearrangement to form a
more stable ketoamine (HbA1c) (Hare, Shaw and Zimmet, 2012). As seen in figure
1, the two extra products i.e., the formation of intermediate and advanced glycosylation
products can be produced if diabetes is not diagnosed and treated on time.
These extra products are considered to cause further complications for
diabetics as there will be a high risk of retinopathy and neuropathy (Singh
et al., 2014).   


addition to HbA1c, as known that Red blood cells carry oxygen
to the tissues through the blood flow of the circulatory system, they contain
haemoglobin and when the haemoglobin binds with glucose in the blood over the time
of 4 – 12 weeks, it becomes glycated.  This
means that with more glucose present in
the blood, it is more likely that the glucose will interact with the haemoglobin
and make more glycated haemoglobin. This formation decreases oxygen carrying capacity and limits the
tissue oxygen delivery (Kilpatrick, 2000).
Therefore, HbA1c is carried in order to prevent further damage. HbA1c provides
an average glucose concentration over a period of 8-12 weeks (3 months). As haemoglobin
glycation occurs over the life span of 120 days of red blood cells and because
of this lifespan of erythrocytes, HbA1c is limited to 3 months…

of HbA1c:

There are various assays carried out to measure HbA1c. The principle of all these methods is to detach the
glycated haemoglobin from non-glycated haemoglobin. Unlike other glycated
haemoglobin fractions, HbA1c can be separated easily based on differences in net
charge (usually by HPLC) or structure (usually immunoassays or boronate
affinity chromatography). HPLC is the most commonly used assay for measuring
HbA1c as Hb species are eluted from the exchange column at different rates due to
charge variability between HbA1c and other haemoglobin. Spectrometry is then
used to calculate the concentration of HbA1c. However, interference with HBS
can occur during this method.

Immunoassays is another method
which involves the measurements of HbA1c specifically of antibodies in order to
recognise the structure of the N- terminal glycated (usually first 4 – 10 amino
acids) of the Hb beta chain. This method allows the use of the PCO device,
established by WHO. This device can measure the HbA1c concentration in a
hospital rather than laboratories however, the disadvantage of this assay can
be the variable interference of haemoglobinopathies with altered amino acids on
binding sites.

Boronate affinity method is the less common one, which uses m-aminophenylboronic
acid to react specifically with the cis-diol groups of glucose on Hb. This method measures
total glycated GHB, including HbA1c and Hb glycated at other sites, and tends
to demonstrate the least interference from the presence of Hb variants and
derivatives. The enzymatic method currently available measures HbA1c by using
an enzyme that specifically cleaves the N-terminal valine. Nevertheless, this method
does not only measure the glycation
of N-terminal valine on ? chain, but also ? chains glycated at other sites and
glycated ? chains.

Total GHB is a term used to refer to all glycated Hb species. Total GHB
is composed of HbA1c, and Hb glycated with glucose at other sites, including
the glycated N terminus of the ? chain and the glycated ? amino groups of
lysine residues. It can be measured by affinity chromatographic methods.


in which HbA1c should not be used

For the vast majority of individuals with
diabetes, HbA1c is considered to be an excellent measurement of
the glycaemic control. However, WHO provided a few clinical scenarios in which hbA1c
testing is considered unreliable (summarised in table 2 below). Most commonly,
conditions that alter erythrocytes lifespan, glycation rate and availability of
glucose can significantly affect the accuracy of HbA1c. These conditions may
either result in decreased or increased hbA1c value. When
erythrocyte lifespan is shortened, the HbA1c level is lowered, thus providing a
false negative result but when the lifespan is extended, the HbA1c is
increased, which also gives a falsely positive result (Florkowski, 2013).



Effect on HbA1c

haemolytic anaemia

lowers HbA1c concentration due to the lysis of

Severe iron-deficiency anaemia

causes 1-1.5% rise in HbA1c due to lengthened
erythrocyte lifespan

Pregnant individuals

Lowers hba1c due to extended erythrocyte lifespan

Individuals on medication, which increases glucose
concentration due to insulin resistance e.g. steroids (Ahmed, 2011).

Increases A1c thus would give falsly high hba1c

children and young people under 18 or Type 1
diabetes patients.

Decreases A1c due to rise in plasma glucose therefore,
would give falsely low hba1c.

Any type of
haemoglobinopathies patients

A1c is decreased due to an increased amount of non-HbA haemoglobin
thus would result in low hba1c levels.


Advantages and Disadvantages of

The following table summarises the advantages and disadvantages of using HbA1c.



Can measure chronic glycaemia levels.

Results can
be misleading in certain physiological and disease states

It can be done at any time of the day.

Still not available

HbA1c is not disturbed by temporary lifestyle alterations unlike FPG
and OGTT (Saudek et al., 2008)


HbA1c is more convenient than other glucose tests as there is no
requirements for pre-test preparations such as fasting.

Does not directly
indicate the measurement of glycaemia.








Limitations of HbA1c:

Glycation being not a direct measurement of glycaemia can be
affected by other factors, which would give incorrect hbA1c readings. Factors such as race or age are also reported to
influence HbA1c. For example, the level of HbA1c is higher in
people from Asia and Africa compared to Caucasians. This means that different
cut off points for varying ethnicities must be assigned as individuals could be
under and over diagnosed if the same value is used. Therefore, it is
recommended that an alternative measure of glycaemic control is used in such
circumstances, where HbA1c methods are invalidated.

More research needs to be done in order to provide more accurate,
reliable and variable reference values of HbA1c for various ethnicities and
different ages. Those different values can then be used worldwide to evaluate
successful diagnosis of diabetes Mellitus. 
HbA1c assays needs to be standardised internationally (by following IFCC
and NGSP guidelines) to improve the accuracy of the diagnosis of diabetes