Biochemical investigation, diagnosis

and monitoring of DM. HbA1c –

methodology, Hb variants, target
levels and standardization
SYLVIA ACHIENG LUMUMBA
2021/MMLS/016/PS
LEC: MS KICONCO RITAH
Biochemical investigation, diagnosis and
monitoring of DM
• Diabetes mellitus (DM) is a
chronic metabolic disorder
characterized by high blood
sugar levels due to either
• Insufficient insulin production
–type I DM
• Inability of the body to use
insulin effectively- Type II DM
• Biochemical investigations
are essential for the diagnosis
and monitoring of DM.
Serum biomarkers of DM type I
Biomarkers of Diabetes Melitus typeII
Biochemical investigation, diagnosis and
monitoring of DM
There are several categories of DM, including:
• Type 1 diabetes: This is an autoimmune disease in which the body's
immune system attacks and destroys the insulin-producing beta cells
in the pancreas.
• Type 1 diabetes usually develops in childhood or adolescence, and
people with this condition require lifelong insulin therapy.
• The diagnosis of type 1 diabetes is made based on clinical symptoms,
such as polyuria (excessive urination), polydipsia (excessive thirst),
and weight loss, and confirmed by measuring blood glucose levels and
detecting autoantibodies against pancreatic beta cells.
Biochemical investigation, diagnosis and
monitoring of DM
• Type 2 diabetes: This is the most common type of diabetes, accounting
for approximately 90% of all cases.
• Type 2 diabetes usually develops in adulthood and is characterized by
insulin resistance, which means that the body's cells are less responsive to
insulin.
• As a result, the pancreas produces more insulin to compensate, but
eventually, it may not be able to keep up with the demand, leading to high
blood glucose levels.
• The diagnosis of type 2 diabetes is made based on clinical symptoms,
such as polyuria, polydipsia, and fatigue, and confirmed by measuring
blood glucose levels.
Biochemical investigation, diagnosis and
monitoring of DM
• Gestational diabetes: This is a type of diabetes that develops during pregnancy
and usually resolves after delivery.
• Gestational diabetes is caused by hormonal changes that lead to insulin
resistance.
• Women who develop gestational diabetes are at increased risk of developing type
2 diabetes later in life.
• The diagnosis of gestational diabetes is made by performing a glucose tolerance
test during pregnancy.
• Other types of diabetes: There are several other types of diabetes that are less
common, including genetic forms of diabetes, drug-induced diabetes, and
diabetes associated with other medical conditions.
Biochemical investigation, diagnosis and
monitoring of DM
• When diagnosing diabetes in the general population, it is most
common to detect and evaluate hyperglycemia and blood glucose-
related markers.
• In combination with AAb testing, T1D can be typically differentiated
from other forms of diabetes.
• TYPES of biomarkers
• Blood glucose-related markers
• Autoantibody-based biomarkers
• C-peptide
 Blood glucose-related markers

Fasting Plasma Glucose (FPG) Test

• This test measures the glucose level in the blood after an overnight
fast of at least 8 hours.
• A level of 126 mg/dL or higher on two separate occasions indicates
diabetes.
Oral Glucose Tolerance Test (OGTT)
• This test measures the glucose level in the blood after drinking a
sugary solution.
• A level of 200 mg/dL or higher after 2 hours indicates diabetes
Blood glucose-related markers

Glycated Hemoglobin (HbA1c) Test

• This test measures the average blood glucose level over the past 2-3
months.
• A level of 6.5% or higher indicates diabetes.
Random Plasma Glucose Test
• This test measures the glucose level in the blood at any time of the
day.
• A level of 200 mg/dL or higher along with symptoms of diabetes
indicates diabetes.
Blood glucose-related markers

• Urine Tests: Urine tests may be performed to check for the presence
of glucose and ketones in the urine, which are indicators of
uncontrolled diabetes.
• Lipid Profile Test: This test measures the levels of cholesterol and
triglycerides in the blood, which are often elevated in individuals with
diabetes.
• Kidney Function Test: Diabetes can affect the kidneys, so kidney
function tests may be performed to assess their function.
Biochemical investigation, diagnosis and
monitoring of DM
• In addition to the tests, regular monitoring of blood glucose levels is
essential for individuals with diabetes.
• Self-monitoring of blood glucose (SMBG) is a simple test that can be
performed at home using a glucose meter.
• The frequency of SMBG varies depending on the type of diabetes and
the treatment regimen.
• Overall, biochemical investigations play a crucial role in the diagnosis
and monitoring of DM, and regular monitoring of blood glucose levels
is essential for effective management of the condition.
Autoantibody-based biomarkers

• Autoantibody-based biomarkers Of the many types and subtypes of diabetes only 5-

10% are classified as T1D.
• They usually can be differentiated from type 2 diabetes (T2D) and other subtypes
based on the presence of specific AAbs.
• There are five commonly tested AAb markers used in the diagnosis of T1D which
include
• ICA (isletcell cytoplasmic AAb),
• GADA (glutamic acid decarboxylase (GAD) AAb),
• IA-2A (insulinoma 2 (IA-2)associated AAb),
• IAA (insulin AAb),
• ZNT8A (zinc transporter 8 AAb).
• At least 1 autoantibody is present in > 95% of individuals with T1D upon
hyperglycemia.
C-peptide
• C-peptide Insulin is first synthesized as preproinsulin which becomes
proinsulin after cleavage of the signal peptide in the endoplasmic reticulum.
• Proinsulin is packaged into vesicles and is then cleaved into the active insulin
hormone and C-peptide and both insulin and C-peptide are released in
equimolar amounts from mature granules, together with small amounts of
uncleaved proinsulin.
• Due to hepatic uptake, insulin has a much shorter half-life time (3-5 min)
compared to proinsulin and C-peptide.
• Stimulated serum C-peptide level (as a surrogate for insulin) has been
considered a consistent and sensitive measure of beta-cell function and can be
used to help differentiate autoimmune-diabetes from other diabetes subtypes.
C-peptide
• C-peptide test needs to be combined with other factors, such as BMI, for
better interpretation of disease progression.
• C-peptide measurement is also currently the most suitable primary outcome
for clinical trials of therapies aimed at preserving or improving endogenous
insulin secretion in T1D patients.
• However, C-peptide measurement may not be as reliable with the increasing
obesity epidemic.
• The manifestation of clinical T1D represents the near end-stage of beta-cell
destruction since only 10–20% of the insulin producing β-cells have been
estimated to still be functioning at the time of diagnosis.
• Therefore, it is imperative to have specific markers that can monitor and
predict the disease progression.
HbA1c – Methodology
• HbA1c is a blood test that measures the average blood sugar level over the past
2-3 months in individuals with diabetes.
• The HbA1c test principle: that glucose molecules can attach themselves to the
hemoglobin protein in red blood cells. This attachment process is known as
glycation, and the resulting compound is called glycated hemoglobin or HbA1c.
• It monitors long-term glycemic control in individuals with diabetes.
• The higher the level of glucose in the blood, the more hemoglobin becomes
glycated.
• The test measures the percentage of glycated hemoglobin in the blood.
• The normal range is below 5.7%. For people with diabetes, the target range for
HbA1c is usually between 6.5% and 7.5%.
Methods for measuring HbA1c
High-Performance Liquid Chromatography (HPLC)
Immunoassay
Capillary Electrophoresis
Point-of-Care Testing
High-Performance Liquid Chromatography (HPLC)

• In the first step haemoglobin is cleaved into

peptides by the proteolytic enzyme
endoproteinase Glu-C.
• Thereafter the resulting glycated and non-
glycated Nterminal hexapeptides of the β-
chains are separated from the crude peptide
mixture and quantified by HPLC and
electrospray mass spectrometry or by HPLC
followed by capillary electrophoresis with UV
detection.
• The percentage of HbA1c is determined as a
ratio of the glycated to non-glycated β-N-
terminal hexapeptides of haemoglobin.
Steps into HbA1c test using HPLC
Step 1; Isolation of erythrocytes and haemolysis
• Human erythrocytes are separated, washed and haemolysed in water and kept in a
storage buffer at pH 6.2.
Step 2; Enzymatic cleavage of the haemoglobin
• The haemoglobin solution is treated with endoproteinase Glu-C
• The protein is hydrolised into several peptides, among them the glycated (HbA1c) and
the non-glycated (HbA0) N-terminal hexapeptides of the β-chains.
Step 3; Analysis and calibration
• The peptide mixture is analysed to measure the ratio of glycated to non-glycated
hexapeptide.
• Calibrators consisting of the mixture of pure HbA1c and HbA0 are used for calibration.
HbA1c result interpretation

Note; diagnosis of T2DM in asymptomatic

individuals requires two results ≥48
mmol/mol (≥6.5%). If the second sample
is <48 mmol/mol (<6.5%) the person
should be treated as at high risk of
diabetes.
Methods for measuring HbA1c
Immunoassay
• This method uses antibodies to specifically detect and measure HbA1c
in a blood sample.
• The blood sample is mixed with antibodies that bind to HbA1c, and
the resulting complex is measured using a machine.
• The results are reported as a percentage of the total hemoglobin.
Methods for measuring HbA1c
Capillary Electrophoresis
• This method uses an electric current to separate HbA1c from other
forms of hemoglobin in a blood sample.
• The separated HbA1c is then measured using a machine.
• The results are reported as a percentage of the total hemoglobin.
Methods for measuring HbA1c
Point-of-Care Testing
• This method uses a portable device to measure HbA1c in a fingerstick
blood sample.
• The results are available within a few minutes and can be used for
immediate clinical decision-making.
 Interference of HbA1c test
Carbamylated and acetylated haemoglobin
• Carbamylation and acetylation can also modify the Nterminal valine that is the major glycation
site. These compounds interfere with many ion exchange chromatography methods
• Carbamylation occurs to a much higher degree in patients with reduced kidney function and
elevated serum urea levels than those with kidney function.
• A normal sample was carbamylated in vitro by potassium cyanate (1 mmol/l, for 60 min at 37
°C). HbA1c increased from 5.2 to 5.9 measured by Mono S ion exchange chromatography but
the HPLC-CE procedure gave identical results.
• Some patients on high dose of aspirin show small amount of acetylated haemoglobin.
• Normal HbA1c samples were therefore incubated with acetylsalicylic acid, 5 mmol/l, for 30
min at 37 °C. The HbA1c increased from 5.2 to 5.5%.
• The HPLC-CE results were the same before and after acetylation. Thus no interference was
shown.
Effects of Hb variants on HbA1c
• The accuracy of the HbA1c test can be affected by several factors,
including hemoglobin (Hb) variants.
• More than 750 different haemoglobin variants exist.
• Hb variants are genetic mutations that affect the structure of the
hemoglobin protein, which can lead to inaccurate HbA1c test results.
For example
• Hb S with the amino acid substitutions of glutamic acid to valine
• Hb C with glutamic acid to lysine, respectively at position 6 from the
N-terminal end.
Sickle cell anemia(HbS)

• Sickle cell anemia is a genetic disorder that cause the red blood cells
to become rigid and sickle-shaped.
• Its caused by a mutation in the beta-globin gene to produce an
abnormal type of hemoglobin hemoglobin S (HbS)
• The presence of HbS can lead to lower HbA1c levels due to the
shorter lifespan of HbS in the blood.
• The longer a red blood cell lives, the more glucose it can bind to,
resulting in higher levels of HbA1c.
Hemoglobin C disease (HbC)
• This disease cause an abnormal version of hemoglobin called HbC.
• The abnormal HbC protein causes the red blood cells to take on a
characteristic crescent or "C" shape.
• Similar to sickle cell anemia, individuals with hemoglobin C disease may
experience complications such as anemia, jaundice, and a higher risk of
infections.
• HbC lowers the levels of HbA1c, which is a marker of long-term blood
sugar control.
• The abnormal HbC protein interferes with the glycation process that leads to
the formation of HbA1c even if their blood sugar levels are poorly
controlled.
Thalassemia

• It is a group of genetic disorders that result in the production of less and

abnormal forms of hemoglobin.
• The effect of thalassemia on blood glucose control is an increase in HbA1c
levels. This is because thalassemia can lead to
• production of abnormal hemoglobin variants that have a shorter lifespan in the
blood compared to normal hemoglobin.
• This can result in increased levels of glycated hemoglobin in the blood, as
the abnormal hemoglobin is more likely to be glycated
• HbA1c levels vary depending on the severity and type of thalassemia.
• Factors such as anemia, iron overload, and chronic inflammation may also
affect blood glucose control in individuals with thalassemia.
Hemoglobin E (HbE)

• This is a genetic variant of hemoglobin that is relatively common in

Southeast Asia, particularly in individuals of Thai, Cambodian, and
Laotian descent.
• HbE is caused by a genetic mutation that affects the production of beta-
globin, one of the two types of protein chains that make up hemoglobin.
• The presence of HbE in the blood can interfere with the binding of
glucose to hemoglobin, resulting in falsely low HbA1c levels.
• This is because HbE has a slightly different structure than normal
hemoglobin, which can affect the binding of glucose to the hemoglobin
molecule.
 Fructosamine test in individuals with
hemoglobin variants
• This is a blood test that measures the level of fructosamine.
• Fructosamine is a compound formed when glucose in the blood binds to
proteins in red blood cells and other tissues.
• Fructosamine testing provides an indication of blood glucose control over
the past 2-3 weeks.
• The test is particularly useful in individuals with
• Hemoglobin variants, such as sickle cell anemia or hemoglobinopathies,
• Conditions that affect the lifespan of red blood cells, such as anemia or recent blood
transfusions,
• Diabetes who have recently started treatment or changed their treatment regimen.
• It provides a more rapid indication of the effectiveness of treatment
changes than HbA1c.
Continuous glucose monitoring (CGM) in
individuals of hb variants
• This is a method of monitoring blood glucose levels continuously
throughout the day and night using a sensor that is placed under the
skin in the interstitial fluid
• The glucose levels are then transmitted to a monitor or smartphone
application, allowing for real-time tracking of blood glucose levels.
• CGM provides a more detailed picture of blood glucose control over
time, including fluctuations in blood glucose levels throughout the day
and night, which can be missed by intermittent blood glucose testing.
Continuous glucose monitoring (CGM) in
individuals of hb variants
• Fructosamine testing, which measures glucose binding to proteins
other than hemoglobin, may be affected by hemoglobin variants,
resulting in inaccurate blood glucose control assessments.
• CGM overcome the limitations by providing real-time data on blood
glucose control, allowing for more accurate management of diabetes
and other conditions that affect blood glucose levels.
• CGM can also be useful in monitoring blood glucose control in
individuals who are undergoing changes to their diabetes management
regimen, such as changes to medication or lifestyle modifications,
allowing for rapid adjustments to treatment as needed.
The target HbA1C levels
• The target HbA1c level for individuals with diabetes is an important goal in
diabetes management, as it reflects the average blood glucose levels over the
previous 2-3 months.
• The target level varies depending on several factors, including
• Age,
• Duration of diabetes,
• Presence of other medical conditions, and
• Overall health status.
• The American Diabetes Association (ADA) recommends a target HbA1c level of
less than 7% for most individuals with diabetes.
• This target level is based on clinical studies that have shown that maintaining
HbA1c levels below 7% can reduce the risk of long-term diabetes complications.
The target HbA1C levels
• HOWEVER in older adults or individuals with a history of severe
hypoglycemia, a higher target HbA1c level may be recommended to
reduce the risk of hypoglycemia,
• Individuals with a shorter duration of diabetes or a lower risk of
complications, a lower target HbA1c level may be recommended
• Note that the target HbA1c level should be individualized based on a
comprehensive assessment of an individual's medical history, current
health status, and diabetes management goals.
• Regular monitoring of blood glucose levels and HbA1c levels can help
individuals and healthcare providers adjust treatment plans as needed to
achieve and maintain target blood glucose levels.
Standardization of the HbA1c test
• Standardization is essential to ensure accuracy and consistency of test results
across different laboratories and testing methods.
• The standardised non-diabetic reference range is 20 – 41 mmol/mol in IFCC
units or 4.0 – 5.9% in DCCT units.
• The International Federation of Clinical Chemistry and Laboratory Medicine
(IFCC) and the National Glycohemoglobin Standardization Program (NGSP)
have developed a standardized method for HbA1c testing, which allows for
comparison of test results across different laboratories and methods.
• The NGSP uses a standard reference method that is traceable to the Diabetes
Control and Complications Trial (DCCT) to ensure accuracy and consistency
of HbA1c test results.
Standardization of the HbA1c test
• The IFCC has developed a standardized reference measurement procedure
for HbA1c, which provides a universal method for calibrating HbA1c testing
methods and ensuring consistency across different testing platforms.
• The NGSP uses a standard reference method that is traceable to the Diabetes
Control and Complications Trial (DCCT) to ensure accuracy and consistency
of HbA1c test results.
• The DCCT was a large clinical trial that demonstrated the relationship
between blood glucose control and the risk of diabetes complications.
• The reference method used by the NGSP is based on high-performance liquid
chromatography (HPLC) and provides a standardized HbA1c value that can
be used to calibrate other testing methods and ensure accuracy of test results.
Standardization of the HbA1c test
• In addition to the standard reference method, the NGSP also provides
a certification program for HbA1c testing laboratories.
• Laboratories that participate in this program are required to
demonstrate accuracy and precision of their testing methods and
undergo regular proficiency testing to ensure consistency of test
results.
Dual unit reporting of HbA1C
• This refers to reporting the test results in both IFCC units (mmol/mol) and DCCT units
(%).
• Dual unit reporting is particularly useful when patients move between countries or when
healthcare providers need to compare results from different laboratories that use different
units of measurement.
• It can also be helpful for patients who are used to one unit of measurement and may find it
easier to understand their results if they are reported in both units.
• The formula for converting IFCC units to DCCT units is DCCT %
• = (IFCC mmol/mol - 2.15) x 10.929,
• The formula for converting DCCT units to IFCC units is IFCC mmol/mol
• = (DCCT % / 10.929) + 2.15.
• These formulas take into account the different ways in which HbA1c is measured and
expressed in the two different systems.
Refrences
• American Diabetes Association. Standards of medical care in diabetes
—2021. Diabetes Care. 2021;44(Suppl 1):S1-S232.
• International Federation of Clinical Chemistry and Laboratory
Medicine (IFCC). HbA1c Standardization. Available at:
https://www.ifcc.org/ifcc-scientific-division/sd-working-groups/clinica
l-applications-of-c-reactive-protein-crp-and-cardiovascular-risk-ifcc-c-
rp-wg/hba1c-standardization/
. Accessed on March 28, 2023.
• Little RR, Rohlfing CL, Sacks DB. Status of HbA1c measurement and
goals for improvement: from chaos to order for improving diabetes
care. Clin Chem. 2011;57(2):205-214.