Centrifugation is used to separate blood components by density. Plasma, which makes up 55% of blood volume, is the pale liquid found at the top after centrifugation. It contains water, proteins, electrolytes, nutrients, waste products, and gases. The two major classes of plasma proteins are albumin and globulins. Albumin maintains colloid osmotic pressure and transports molecules like hormones, fatty acids, and drugs throughout the body. Low albumin can cause edema. Globulins include immune proteins and transport proteins. Common plasma tests measure total protein, albumin, globulin, and albumin/globulin ratios to assess health conditions.

1. Plasma Protein
By
N.Santhosh Kumar
Asst.Professor
Department of Biochemistry
SIMS & RH

2. SEPARATION OF BLOOD COMPONENTS BY
CENTRIFUGE
•The formed elements like cells can be separated from plasma by centrifuge.
•Blood sample is spun for a few minutes in a tube to separate, its
components according to their densities.
Blood
↓
Place in suitable tubes
↓
Centrifuge with
appropriate rpm
↓
Obtain Components
BLOOD COMPONENTS
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

3. Plasma:
•Plasma makes up the top of the tube, which is a pale red color and
contains 55% of the total volume.
Buffy coat:
• It is the narrow cream colored found above the RBCs formed by the
WBCs and platelets.
Hematocrit:
•Volume when RBCs settle to the bottom of the test tube which is
about 45% of the total volume, also called packed cell volume (PCV)
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

4. 55%
45%
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

5. • Serum and plasma both come from the liquid portion of the blood that
remains once the cells settle down after centrifugation
PLASMA SERUM
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH
Centrifugation plasma obtained by centrifuging
blood
no need centifuge
Anticoagulants Needed Not needed
Fibrinogen Present Absent

6. PLASMA:
• Plasma is a clear, colored liquid portion of the blood.
Composition
Components Functions
Water (91.5%) Liquid portion of blood.
Acts as solvent and suspending medium for components of blood.
Plasma proteins
(7%)
Exert colloid osmotic pressure, which helps to maintain water balance
between blood & tissues and regulates blood volume
Other solutes
(1.5%)
Na+, K+, Ca+, Mg2+, Cl-, HPO4
-, SO-
4, HCO-
3 are inorganic salts
Nutrients- amino acids, glucose, fatty acids, vitamins and minerals
Oxygen, carbon dioxide & nitrogen are gases
Regulatory substances such as enzymes, hormones, vitamins
Waste products- urea, uric acid, creatinine, bilirubin and ammonia etc,
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

7.  Major plasma proteins - Albumin
Globulin
Fibrinogen
 The human plasma proteins are a mixture of
Simple proteins – Albumins
Conjugated protein – Lipoprotein & Glycoprotein
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

8. Separation of plasma proteins
Salting out
Electrophoresis
Immunoelectrophoresis
Ultracentrifugation

9. Functions of plasma proteins
• Maintain colloidal osmotic pressure of blood
• Maintain viscosity and blood pressure
• Essential for blood clotting
• Transport of certain substances in blood
• Involved in Erythrocyte sedimentation rate (ESR)
• Act as buffers
• Act as a protein reserve
• Antibodies
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

10. TOTAL PROTEINS
It represents the sum of albumin and globulins
Normal levels is 6 to 8 gm%
•Increased in chronic infection, liver dysfunction & alcoholism,
dehydration, hemolysis & leukemia
•Decreased in malnutrition and malabsorption, liver disease,
diarrhea & severe burns
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

11. ALBUMIN
• Simple, globular & heat coagulable protein with mol wt is 69
kDa and isoelectric pH is 4.7
• Synthesized in liver (10-15g/day) & has half life is 20 -25 days.
• Consists of a single polypeptide chain having 585 a.a’s with 17
intrachain disulfide linkages.
• Precipitated by full saturation with ammonium sulphate.
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

12. Functions of Albumin
1. Maintain colloidal osmotic pressure in vascular & extra vascular
spaces
2. Max buffering capacity (histidine)- maintain acid-base balance
3. Transport of
4. It exerts low viscosity.
5. First class protein - tissue proteins, enzymes & hormones etc.
Metabolites(Bilirubin)
Steroid
hormones
Metabolic waste
Nutrients(Fatty acids)
Drugs (Sulfonamides, Penicillin, Aspirin)
Ca++
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

13. Clinical significance of Albumin
(Normal range-3.5 - 5 gm%)
Hypoalbuminemia
Decreased conc. of
alb in serum.
- Cirrhosis of liver,
- Nephrotic syndrome,
-Malnutrition,
-Burns (-ve nirogen balance) and
-Protein losing enteropathy
Analbuminemia
(less than 1gm%
Congenital rare autosomal recessive
disorder
Defective mutations in the gene is
responsible for absence of albumin
synthesis.
A/G ratio is reversed
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

14. Clinical significance of Albumin
Albuminuria
Albumin is excreted
into urine(30mg/day)
-Renal diseases
-Nephrotic syndrome,
-Diabetic Nephropathy,
-hyper tention
-Certain inflammatory conditions of urinary
tract.
Micro albuminuria
(about 30-300mg/ day)
In renal disease &
Diabetic Nephropathy
Decreased in plasma albumin levels (fall in osmotic pressure) &
excreted in urine leads to edema
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

15. Role of albumin in oedema (Oncotic pressure)
• OP/COP is a form of Osmotic pressure, Induced by albumin in blood vessel’s
plasma
• Total COP is 25mmHg (75-85% is contributed by albumin)
• COP of the blood is imp in maintaining a balance b/w the water inside the blood
& that in the tissue fluid around the cells
• Effective osmotic pressure (EOP) - maintenance of blood volume and
distribution of body fluids
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

16. Pedal edema
• Albumin and other proteins in the blood act
like sponges to keep fluid in blood vessels.
• Low albumin levels leads to decrease in
osmotic pressure, preventing the return of water
into the vesicular compartment –interstitial
oedema
• Pedal edema (accumulation of fluid in the feet
& lower legs).
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

17. Hypoalbuminaemia
(less than 2gm% )
Increased capillary
permeability
Protein loss
- Nephrotic syndrome
- Protein-losing enteropathy
Reduced albumin
synthesis
- Liver disease
- Malnutrition
(Kwashiorkar)
Burns
Trauma
Inflammation or sepsis
Allergic reactions
Adult respiratory distress
syndrome
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

18. N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

19. MECHANISMS
• Venous edema, caused by increased capillary filtration and retention of
protein-poor fluid from the venous system into the interstitial space.
• Lymphatic edema, caused by obstruction or dysfunction of lymphatic
outflow from the legs resulting in accumulation of protein-rich interstitial
fluid.
• These two mechanisms can operate
independently or together
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

20. DURING PREGNANCY
1) Body releases hormones that encourage fluid retention, and a woman
tends to retain more sodium and water than usual- face, hands, lower
limbs, and feet may swell.
2) Resting in a reclined position during pregnancy, the enlarged uterus can
press on inferior vena cava- obstruct the femoral veins
3) The blood clots more easily during pregnancy, can increase the risk of
deep venous thrombosis (DVT)
4) Eclampsia, results from pregnancy-induced hypertension, or high
blood pressure
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

21. Clinical Importance of Albumin
Hyper-albuminemia
Acute dehydration In Vomiting, diarrhoea. No clinical
significance
Hyper-proteinemia
Excessive production
of gamma globulins
- Hypoalbuminaemia
- Multiple myeloma
- Waldenstrom’s macroglobulinemia
↑ Beta globulins - Hyper-lipoproteinaemia
- Other Hyper-lipidaemic conditions
↑Alpha globulins - Nephrotic syndrome
(Alpha 2-macroglobulin is increased)
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

22. GLOBULINS
• Glycoprotein with mol wt – 90 -1300kDa.
• There are separated by half saturation with ammonium sulphate.
• Functions: Transport &Immunity.
• Globulins contributes different fractions in electrophoresis (α1,α2,β, γ
-globulins).
• Normal range of Globulins -1.8 to 3.6 gm%
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

23. Fraction Examples c (gm%)
1-globulins 1-antitrypsin, 1-acid glyco- protein,
RBP, 1-lipoprotein (HDL), 1-
fetoprotein,
0.1 to 0.4
2-globulins Haptoglobin, Ceruloplasmin
2-Macroglobulin, Thyroxin-binding
globulin (TBG),
0.4 to 0. 8
-globulins C-reactive protein (CRP) Transferrin,
Hemopexin, 2-Micro globulin, -
lipoprotein (LDL)
0.5 to 1.2
-globulins IgG, IgA, IgM, IgE, IgD, 0.7 to 1.5
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

24. Name of the
Globulin
Function Normal
range
Clinical significance
Increased in Decreased in
α1 –anti
trypsin
 Inhibits serine protease
activity
75 to 200
mg/dl
Liver disease,
Burns & trauma
Nephrotic
syndrome ,
Emphysema
α1-feto
globulin
 Immuno-regulator
during pregnancy
less than 1
μg/ dl
pregnancy, open
neural tube defect
Hepatoma
Down’s
syndrome
α2-macro
globulin
 Inhibits protease
activity
 Anti coagulant.
 Carrier of many
growth factors
130 to 300
mg/ dl
Nephrotic
syndrome
Acute
pancreatitis ,
Advanced
carcinoma of the
prostate
Globulin Fractions And Its Role In The Body
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

25. Haptoglobin  prevents the loss of
free Hb from the
kidney
40 -170
mg/dl
Inflammatory
conditions
Rheumatic
diseases,
intravascular
haemolytic
anaemia
Ceruloplasmin  Transports copper
ions (6 to 8 cu/ mol).
 Ferroxidase
activities.
 Antioxidant in
plasma
25 to50
mg/dl
Inflammatory
conditions &
Collagen
disorders
Nephrotic
syndrome,
Wilson’s disease
and
Menke’s
diseases
Name of the
Globulin
Function Normal
range
Clinical significance
Increased in Decreased in
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

26. γ- globulins
 γ- fraction of globulins are
immunoglobulin / antibodies.
 Produced by plasma cells
Functions
 To protect against infectious
agents or their products.
.

27. Neutralize toxins & viruses
Opsonize microbes
Activate complement & prevent the
attachment of microbes to mucosal surfaces
Igs provide resistance because they can

28. FIBRINOGEN
 Soluble glycoprotein with mol wt – 3,40,000
 It constitutes 2 to 3% of total plasma proteins.
 Imp soluble plasma clotting factor precursor, participates in blood
coagulation.
 Which converted to fibrin, which polymerizes to form fibrin clot.

30. Clinical significance:
Plasma conc. of fibrinogen – 0.2 - 0.4 gm%
• High fibrinogen levels: inflammatory diseases (rheumatoid arthritis)
• Low levels of fibrinogen: in Liver diseases, bone lesions,
malnutrition &bleeding disorders
N.Santhosh Kumar /Asst.Professor of
Biochem / SIMSRH

31. A/G (ALBUMIN/GLOBULIN) RATIO
• Normal Ranges is 1.2:1 to 1.5: 1
• A/G ratio elevated in hypothyroidism, high protein / high
carbohydrate diet with poor nitrogen retention and Hypo- γ-
globulinemia
• Decreased in liver dysfunction
N.Santhosh Kumar /Asst.Professor of
Biochem / SIMSRH

32. ABNORMAL PLASMA PROTEINS
Bence – Jones proteins Cryoglobulins
Occurs in blood & urine of people
suffering from a disease called
multiple myeloma.
B.J protein have mol wt – 20,000 to
40,000.
Excess synthesed light chain ( k &
λ) of Ig in blood or urine causes renal
tubules damages.
These proteins are coagulated when
plasma or serum is cooled to very low
temp.
It’s mol wt 1,65,000 – 6,00,000.
Increased in rheumatoid arthritis,
Lymphocytic leukemia, Multiple
myeloma
Lymphosarcomas.

33. Next PPT on
PP-02: Globulins, Acute phase proteins, Transport
proteins & Separation Of Plasma Proteins by
Electrophoresis
N.Santhosh Kumar /Asst.Professor of Biochem / SIMSRH

34. Believe In Yourself And
All That You Are.
Know That There Is Something Inside
You
That Is Greater Than Any Obstacle.
Thank You