Determination of Serum/Plasma Proteins
Serum Proteins
Types of Samples
• Plasma
Water + solids (e.g. glucose, urea, albumin, fibrinogen)
No cells
• Serum
Serum = plasma – clotting factors
Plasma vs. Serum
Plasma
Serum
- Anticoagulant
- No anticoagulant
- Contains clotting factors
- Advantage: time saving
- Contains no clotting factors
- Advantage: less interference
Preparation of sample: Plasma
Plasma
anticoagulant
Add venous blood
Mix then centrifuge
Transfer the
clear
supernatant
to specimen
tube
cells
Cent. tube
Preparation of sample: Serum
Serum
Venous
Blood
Allow blood to clot
(20min.)
Remove the clot
and centrifuge
Transfer the
clear
supernatant
to specimen
tube
cells
Cent. tube
Determination of serum proteins
Requirements:
Automatic pipettes
Tips
Cuvettes
 Spectrophotometer
 Reagent
Specimen:
Serum, heparinised or EDTA-plasma
Determination of serum proteins
BIURET METHOD
Principle
Cupric ions complex with the groups involved in the peptide
bond. In an alkaline medium and in the presence of at
least 2 peptide bonds, a violet- colored chelate is formed
Methods:
1- Manual
2- Kit
Determination of serum proteins
Procedure
Blank
Standard
Sample
Determination of serum proteins
Procedure (kit)
Reagents
Blank
Standard
Sample
Distilled water (ml)
50
-
-
Standard (ml)
-
50
-
Sample (ml)
-
-
50
Biuret reagent (ml)
2
2
2
Procedure
Add the reagents and sample as directed
Mix and let stand at room temperature for 10 min.
Read the absorbance at 540 nm.
Zero the spectrophotometer with the blank reagent.
Read the absorbance's of the standard and samples
within 30 min.
Enter the absorbance's readings and calculated the
concentration of the sample.
Determination of serum proteins
Calculation:
Total proteins (g/dl)= Ab. of the sample x conc. of st.(7)
Ab. of the standard
Normal values
Reference Value:
New Born: 5.2-9.1 g/dl
Children: 5.4-8.7 g/dl
Adults:
6.7-8.7 g/dl
Clinical significance
Hyperproteinemia
Definition
An increase in total protein concentration in serum, above
normal
Causes
Dehydration
Asthama
Mineral deficiencies
Allergies
Heart Disease
Clinical significance
Hypoproteinemia
Definition
A decrease in total protein concentration below the reference
causes
Renal diseases
Bleeding
Malabsorption
Malnutrition
Burns
High fever
Liver diseases