ESTIMATION OF PLASMA
GLUCOSE
Rafika Shaikh
Tutor, Dept of Biochemistry
AIIMS Kalyani
Introduction
Blood glucose is the body`s chief source of energy.
Abnormalities of blood glucose level may indicate disturbances of
carbohydrate metabolism.
Diabetes Mellitus is the commonest disease of carbohydrate
metabolism.
The estimation of blood glucose is routinely done for diagnosis and
follow up of Diabetes Mellitus patients.
Importance of Measuring Plasma Glucose
Diagnosis & Monitoring of Diabetes
Gestational Diabetes Mellitus
Preventing long-term complications
Guiding Dietary & lifestyle Choices
Prevalance of Diabetes in India
2021(IDF)
537 million or 10.5%
Global Adult Population
is Diabetic
783 Million or 12.2%
are expected to have
diabetes by 2045
Regulation
of Blood
Glucose
Insulin and
Glucagon
are hormones that
help regulate the
blood sugar levels
in your body.
Biochemical Tests done for Diabetes Assesment
Estimation of Blood Glucose
(Enzymatic/Non- Enzymatic Method)
Glucose Tolerance Test(GTT)
Glycated Hb - HbA1c
(HPLC Method)
Methods Available
Non Enzymatic Methods Enzymatic Methods
1. Nelson-Somogyi method 1. Hexokinase method
2. Folin & Wu method 2. Glucose oxidase Peroxidase
method
3. Asatoor king method
3. Glucose dehydrogenase
4. O-toluidine method method
Sample Collection & Preservation
• Whole blood
• Serum/Plasma
• CSF/Pleural/Ascitic Fluid
• Other Fluid
Sodium Fluoride & Potassium Oxalate (3:1)
Sodium Fluoride is used extensively as a preservative for glucose in
blood samples as it prevents glycolysis by inhibiting the glycolytic
enzyme “ Enolase”
Potassium Oxalate is used as an anticoagulant.
Enzymatic Method of Glucose Estimation
by Glucose Oxidase and Peroxidase Method
(GOD-POD)
Principle:
Read Absorbance
at 490-550nm
Reagents Required:
Phosphate Buffer; pH 7
Glucose Oxidase
Working
Peroxidase Glucose
Reagent
4-aminoantipyrine
Phenol
Glucose standard, concentration 100mg/dL
Test Procedure:
Blank Standard Test
Serum/Plasma - - 10uL
Glucose - 10uL -
Standard
Working 1000uL 1000uL 1000uL
Glucose
Reagent
Mix well. Incubate at 37oC for 10 mins.
Measure the absorbance at 490-550nm
Observation:
Absorbance or optical density (O.D) of Blank= …….
Absorbance or optical density (O.D) of Standard = …….
Absorbance or optical density (O.D) of Test = ……
CALCULATION:
Concentration OD of Test – OD of Blank
of Glucose = x Conc of Std
(mg/dL) OD of Standard - OD of Blank
Where Conc of std = 100 mg/dL
RESULT:
In the given unknown blood sample plasma glucose is
found to be …………mg/dL
Unit of measurement of Glucose
In developed countries plasma or serum Glucose
concentration is estimated and expressed in mmol/L
In India old conventional unit mg/dL is still in use for
convenience of the clinicians.
Depending on time of collection:
Fasting blood glucose- overnight fasting for atleast 8 hrs
preceding the measurement.
Post meal or Post Prandial blood glucose-2 hrs after
ingestion of regular meal.
Random blood glucose -Any time of the day.
NORMAL REFERRENCE RANGE
( as per ADA and CDC guidelines )
Fasting Blood Sugar FBS < 110 mg/dL
Random Blood Sugar RBS < 140 mg/dL
( random state)
Post Prandial Blood Sugar PPBS < 140 mg/dL
Clinical Significance
Hyperglycemia
FBS ≥ 110 mg/dL
Hypoglycemia
PPBS ≥ 140 mg/dL
Blood Glucose <60mg/dL
Causes of Hyperglycemia:
Deficiency or under response to insulin-Diabetes Mellitus
(Type I & II)
Hyperactivity of hormones(Hyperthyroidism,
Hyperpituitarism)
Diseases of Pancreas(Pancreatitis, etc)
Causes of Hyperglycemia:
Gestational Diabetes Mellitus
Patient on steroid drugs(Cortisol, Glucocorticoids)
Miscellaneous Conditions (Genetic Diseases, stress)
Causes of Hypoglycemia:
Overdose of Insulin
Tumour in pancreas/ Insulinomas
Endocrinological disorders(Hypothyroidism,
Hypopituitarism)
Causes of Hypoglycemia:
Oral Hypoglycemic agents(Phenformin)
Prolonged starvation
Severe Excercise
Point of Care Testing (POCT)/ Bedside Testing- Glucometer
Principle-
Reflectance
Photometry
Precaution-Glucometer reading should never be used to confirm the diagnosis of Diabetes.
It is only for monitoring of already diagnosed cases.
Glucose Tolerance Test(GTT)
Glucose Tolerance is defined as the capacity of the body to tolerate an extra load of
glucose.
It is the serial measurement of plasma glucose before and after a specific amount of
glucose load is given orally to the patient.
Can be performed by two ways : 1. Oral GTT
2. Intravenous GTT
It is considered to be a more sensitive test than Fasting blood glucose estimation,
however, the glucose tolerance testing is affected by multiple factors which results
in poor reproducibility.
