Diagnosis and monitoring of

diabetes mellitus
Diagnosis of diabetes is made on the basis of
clinical features and laboratory investigations.
A preliminary screening test may identify the
presence of urinary glucose, although this is
not diagnostic of diabetes mellitus.
A patient presenting with symptoms of
diabetes mellitus must have a venous blood
specimen taken and its glucose concentration
determined.
Types of blood glucose tests
1. Fasting blood sugar (FBS) measures blood
glucose after fasting for at least 8 hours. It
often is the first test done to check for
diabetes.
2. 2-hour postprandial blood sugar (2-hour PP)
measures blood glucose exactly 2 hours after
eating a meal.
3. Random blood sugar (RBS) measures blood
glucose regardless of when the person last ate.
Several random measurements may be taken
throughout the day.
 Random testing is useful because glucose
levels in healthy people do not vary widely
throughout the day. Blood glucose levels that
vary widely may indicate a problem. This test is
also called a casual blood glucose test.
4. Oral glucose tolerance test (OGTT) measures
the body's ability to use glucose.
An oral glucose tolerance test is a series of
blood glucose measurements taken after you
drink a sweet liquid that contains glucose. This
test is commonly used to diagnose diabetes
that occurs during pregnancy.
One-step test
The patients need to go to the lab one time for a
2-hour glucose tolerance test.
For this test:
patients DO NOT eat or drink anything (other than
sips of water) for 8 to 14 hours before test
patients will be asked to drink a liquid that
contains glucose (75 g).
Blood drawn before drink the liquid, and again 2
more times every 60 minutes after drink it. Each
time, blood glucose level will be checked.
Allow at least 2 hours for this test.
Two-step test
The patients need to go to the lab one time for a
2-hour glucose tolerance test.
For this test:
patients DO NOT eat or drink anything (other
than sips of water) for 8 to 14 hours before test
patients will be asked to drink a liquid that
contains glucose (100 g).
Blood drawn before drink the liquid, and again 3
more times every 60 minutes after drink it. Each
time, blood glucose level will be checked.
Allow at least 3 hours for this test.
Normal and abnormal value
A fasting blood sugar level less than 100
mg/dL (5.6 mmol/L) is normal.
A fasting blood sugar level from 100 to 125
mg/dL (5.6 to 6.9 mmol/L) is considered
prediabetes.
If it's 126 mg/dL (7 mmol/L) or higher on two
separate tests, means diabetes.
A random blood glucose test, a normal result
depends on when you last ate. Most of the time,
the blood glucose level will be below 125 mg/dL.
A level of 200 mg/dL or higher often means you
have diabetes.
Determination of Glucose
Samples For glucose determination:
Whole blood, Plasma /serum, Urine & Other body
fluids.
There are two groups of colorimetric methods for
the quantitative determination of glucose in
either manual, or automated systems.
1. Chemical Methods
A. Oxidation Reduction method
B. Aromatic amine method
2. Enzymatic Method
1. Chemical Methods
A. Oxidation Reduction method
This method depends on reducing property of
glucose.
In Alkaline medium the aldehyde groups in
glucose reduces substance like cupric, ferric,
mercuric salt or other active substances and
produces/reduces color
These reactions are used to measure the
concentration of glucose in biological fluids.
It is a non specific method for glucose and thus
results in falsely increased values.
Non glucose reducing substances ( NGRS)
interferes in this method.
Interference from NGRS may be minimized by
using a protein free filtrate( PFF)-most NGRS co
precipitate with the protein.
NGRS includes glutathione( found in RBCs), uric
acid, creatine, creatinine, ascorbic acid, amino
acids, phenols ,glucuronic acid, sugar
phosphates, other monosacharides &
disacharides etc. Therefore, they are not/rarely
used methods in clinical Labs.
There are two groups of methods widely used in
clinical laboratories:
1. Alkaline Copper Reduction Method
Principle: Depends on the reduction of copper (II)
to copper (I) in alkaline solution while glucose is
oxidized to gluconic acid. The copper (I) ion so
formed reacts with arsenomolybdic acid
(phosphomolybdic acid) to form a blue solution.
The intensity of the blue color is directly
proportional to the concentration of glucose
present in the sample and the absorbance is read
spectrophotometrically at 680 nm.
2. Alkaline Ferri-cyanide method
Principle: Depends on the reduction of Iron (III)
which is yellow to Iron (II) which is colorless in
alkaline solution while glucose is oxidized to
gluconic acid.
The decrease in the intensity of the yellow color is
directly proportional to the concentration of
glucose present in the sample and the absorbance
is read spectrophotometrically at 420 nm.
B. Aromatic amine method
Various aromatic amines react with glucose in hot
acetic acid to form colored derivatives.
The aromatic amine commonly used is ortho-
toluidine.

This method is simple and more specific than the

oxidation-reduction methods but o-toluidine is
carcinogenic and thus its use has tremendously
decreased in clinical laboratories
Principle of ortho-toluidine:
The method involves a reaction between
aldehyde group of glucose and the amine group
of o-toluidine to form a green colored derivative,
glucoseamine or Schiff base.
The intensity of the green color is directly
proportional to the concentration of glucose
present in the sample and the absorbance is read
spectrophotometrically at 630 nm
2. Enzymatic Methods
These methods are more specific to glucose than
the chemical methods.

Almost all clinical laboratories today use enzymatic

methods.

There are two groups of methods which uses

different enzymes:
A. Glucose Oxidase Method ( GOD-PAP)
B. Hexokinase UV Method
A. Glucose Oxidase Method (GOD-PAP)
Principle: Glucose is oxidized to gluconic acid by a
specific enzyme, glucose oxidase. When glucose is
oxidized H2O2 is released proportionally.

The formed hydrogen peroxide reacts under

catalysis of peroxidase with phenol and 4-
aminoantipyrine to a red-violet quinoneimine dye
as indicator.
The intensity of the red-violet color is directly
proportional to the concentration of glucose
present in the sample and the absorbance is
read spectrophotometrically at 546 nm.
B. Hexokinase UV Method
This method is less subject to interference than
the glucose oxidase method.
Principle: Hexokinase is an enzyme that catalyzes
the phosphorylation of glucose by ATP to form
glucose-6-phosphate and ADP.
Glucose-6-phosphate dehydrogenase (G6PDH) is
used to catalyze the oxidation of glucose-6-
phosphate by NADP+ to form NADPH in direct
proportion to glucose present in the sample.
NADPH absorbs light at 340 nm.