This is my first upload , this will be more helpful for who is seeking for bioassay in detail.

1. BIOASSAY OF HEPARIN
SODIUM I.P
Presented by :
S. THAARANI
20CQ1S0705
Dept of pharmaceutical analysis
Seven hills college of pharmacy
Tirupati

2. Introduction:
› Heparin is a naturally occurring , highly sulfated polysaccharide,
of natural origin.
› Heparin was discovered by ‘Jay McLean’ in 1916, heparin has
become widely used as an anticoagulant medicine for the
treatment and prevention of blood clots, thrombotic diseases.
› It is obtained from mucosal tissue of animals such as pigs
(porcine), cattle (bovine).
› Further it is prepared as unfractionated heparin (UHF) or low
molecular weight heparin (LMWH).

3. Bioassay of Heparin:
Principle of heparin bioassay
The potency of heparin sodium is determined
by comparing the concentration necessary to prevent the
clotting of plasma with the concentration of standard
preparation of heparin sodium.
Requirements
1. Prepared plasma
2. Standard preparation of
heparin
3. Heparin to be tested
4. Calcium chloride

4. Sodium
citrate
Prepared plasma
The blood is placed in a test-tube containing 8% w/v sodium citrate
( 19:1 ratio for blood to sodium citrate )
Above solution is immediately mixed by agitating gently and inverse the test tube
Then the mixture is centrifuged , plasma is separated out
0.2 ml of 1% w/v of CaCl2 solution is added to 1ml of separated plasma,
This plasma becomes suitable if blood clots within 5 mins.
1.PREPARED PLASMA

5. 2.STANDARD HEPARIN
Standard heparin
Purified freeze dried
heparin sodium
• The potency of standard heparin is determined according to
the International Standard stated by the WHO .
Range- 0.5 to 2 units / ml
STANDARD SOLUTION: Minimum quantity of standard preparation of
heparin sodium, when added in 0.8 ml of saline solution, maintain
fluidity in 1ml of prepared plasma for 1 hr after addition of 0.2 ml of
CaCl2.
3.TEST SOLUTION
• Weight accurately 25 mg of test sample
• Required saline is added to make the concentration of 1 mg/ml
• Dilute test solution corresponds to that of standard

6. METHOD:
In a sterilized test-tubes add solution of standard preparation
( largest dose does not exceed 0.8 ml ).
Required amount of saline is added to make total volume of 0.8 ml,
followed by 1 ml of prepared plasma to each test tubes.
Add 0.2 ml of 1 % w/v of CaCl2 , the time is noted and each test tube is
immediately stoppered with a suitable rubber stopper.
Above contents are mixed by inverting 3 times in a way
the entire inner surface of tube is wet.

7. [ Set up a series of test preparation ]
The sample of heparin to be tested is diluted according
to same concentration corresponding to that of standard.
Complete the entire process within 20 mins
after addition of prepared plasma
1 hr after addition of CaCl2 solution,
determine the extent of clotting in each test tubes
( Recognize 3 GROUPS between zero and full clotting )
Dilution of test and standard shows same degree of clotting.

8. =
=
S1 T1
If the degree of clotting in dilution of standard preparation lies
between that observed in two series of dilution of test preparation ,
the potency of latter is examined
If there is no correspondence between the degree of clotting
{ standard and test } ,
new dilution is prepared and assay is repeated.
The estimated potency of preparation is calculated by combining the result of assay with
Standard stastical method.
Results

9. BIOLOGICAL ACTIVITY OF HEPARIN:
THERAPEUTIC USES OF HEPARIN:
 Anticoagulant
 Hemodialysis
 Extracorporeal circulation such as liver dialysis
 Deep vein thrombosis ( DVT )
 Treatment of varicose veins
Heparin interact and regulate the activity of widerange of
proteins that are needed for Important biological processes such as,
• Blood anticoagulation
• Cell growth and
migration
• Inflammation
• Pathogen infection

10. THERAPEUTIC USES OF HEPARIN :
 Acute MI
 Peripheral arterial diseases
 Arteriosclerosis
 Lung thrombo embolism
CONCLUSION:
Heparin bioassays are carried out to monitor and adjust
standard heparin
And to evaluate the concentration of heparin in blood and to
monitor the therapy

11. BIOLOGICAL TESTS FOR HEPARIN THERAPY:
1) Whole blood clotting time ( WBCT )
2) Activated clotting time ( ACT )
3) Thrombin clotting time ( TCT )
4) Activated partial thromboplastin time ( APTT )