This presentation is on the bioassay of heparin which helps to know the potency of new heparin drug or heparin conc in individual suffering from heparin resistance diseases. This was made by my friend Naailah and me. Hope it helps.

1. Presented by Nabiilah & Naailah
Date: 4th Feb 2015

2. • Introduction on Heparin
– Biological activity of Heparin
– Therapeutical Uses
• Assay of Heparin
– Principle
– Standard Preparations
– Methods
– Results
– Calculations
– Limit of Errors
• Conclusion

3. Introduction
• Heparin is a highly-sulfated
glycosaminoglycan of natural origin.
• It is also one of the oldest drugs still in
widespread use
– Heparin, along with vitamin K antagonists, have
been the main anticoagulant drugs for more than
70 years, as it has been used since the 1930s.
• Although it was first discovered almost a
century ago, many years would have to pass
until it could be mass produced and used as
an anti-coagulant.

4. Biological Activity
• Heparin can interact and regulate the
activities of a wide range of proteins that
are essentials to important biological
processes such as
– blood clotting
– pathogen infection
– cell differentiation
– cell growth and migration
– inflammation

5. Therapeutic Uses
• The general medical uses of heparin are the
following:
– Acute myocardian infarction
– Curative and prophylactic treatment of
arterial and venous thrombo-embolism.
– Lung thrombo-embolism
– Prevention of deep venous and pulmonary
thrombo-embolism during pregnancy
– Peripheral arterial diseases
– Arterioesclerosis
– Extracorporeal circulation

6. Therapeutic Uses (Cont’d)
– Anticoagulant
– Hemodialysis
– Extracorporeal therapies such as heart-lung
oxygenation and liver dialysis
– Open heart surgery
– Deep vein thrombosis
– Vitreoretinal surgery
– External use for ulcer treatment
– External use for treatment of varicose veins.

7. Bioassay of Heparin• The potency of heparin sodium is
determined by comparing the
concentration necessary to prevent the
clotting of plasma with the
concentration of the Standard
Preparation of heparin sodium
necessary to give the same effect
Principle
of heparin
bioassay:
Reagents
required to
perform
bioassay of
heparin
Heparin to
be tested
(synthesised)
Standard
preparation
of heparin
solution
Prepared
Plasma
Calcium
chloride

8. Prepared plasma
The blood is placed into a test-tube with 8 % w/v sodium
citrate (ratio of blood to sodium citrate is 19: 1)
The above is immediately mixed by gentle agitation and
inversion of the vessel
The mixture is centrifuged and the separated plasma is
pooled out.
0.2 ml of 1% w/v solution of CaCl2 is added to 1ml of the
pooled plasma. This plasma becomes suitable if clot forms
within 5 min.
Prepared
plasma
Sodium
citrate

9. •The potency of standard heparin is determined in
relation to the International Standard stated by the
World Health Organization.
Test solution:
•Accurately about 25 mg of the test sample is weighed
•Sufficient saline is added to give the concentration of 1
mg/ml
Standard heparin:
purified freeze-dried
heparin sodium salt
Standard heparin:

10. Method:
In clean test-tubes, graded amount of the solution of standard
preparation is added (the largest dose not exceed 0.8 ml)
Sufficient volume of saline is then added to make total volume of 0.8
ml and add 1 ml of prepared plasma to each test tube
0.2 ml of 1 % w/v solution of calcium chloride is added, the
time is noted and each tube is immediately stoppered with a
suitable stopper
The contents are mixed by inverting three times in such a way
that the entire inner surface of the tube is wet

11. The sample of heparin to be tested is diluted to the same
concentration corresponding to that of the standard
The procedures are repeated as mentioned for the standard heparin
NB: The entire process of preparing and mixing the tubes of both the solution
of standard preparation and the test solution must be completed within 20
minutes after the addition of the prepared plasma
Exactly one hour after the addition of the calcium chloride solution,
the extent of clotting is determined in each tube, recognizing three
grades between zero and full clotting.

12. • If the degree of clotting observed in the series of dilutions
of the solution of standard preparation lies between that
observed in two of the series of dilutions of the sample
being examined, the potency of the latter is estimated
• If there is no such correspondence between the degrees of
clotting produced by the solution of standard preparation
and any of the dilutions of the sample being examined,
new dilutions of the latter are prepared and assay is
repeated
=
S1
T1
≠
Results:

13. • The estimated potency of the preparation being
examined is calculated by combining the results
of these assays by standard statistical methods.
• The ratio of a given reference standard dose to the
corresponding unknown dose is designated by R.
• The logarithm of the ratio of potency of the
unknown, in quantities assumed to be equal to
those of the reference standard, is designated by
M'
Calculations:

14. Limits of errors:
• The Limit of errors of estimated
potency (P=0.99) are in the range of
–90-110 with three determination
–92-108 with four determination

15. Conclusion
• Heparin bioassays are performed to monitor
and adjust standard heparin.
• This is done in order to evaluate the
concentration of heparin in blood and helps
doctors to monitor therapy.
• If concentrations are within an established
therapeutic interval and the person is doing
well clinically i.e. there is no clotting,
excessive bleeding, or other complications –
then the dosage is considered appropriate.

16. References
• http://www.heparinscience.com/therapeutical_
uses.html
• http://www.slideshare.net/ParasuramanParasu
raman/1-bioassay-of-heparin-17-913-
27391284
• http://www.drugs.com/heparin.html
• http://labtestsonline.org/understanding/analyte
s/heparin/tab/test/
• http://www.scielo.br/scielo.php?pid=S1984-
82502012000300024&script=sci_arttext