Bio assay of heparin

1. BIO ASSAY OF HEPARIN
Submitted By
K. Jayalakshmi
D/o Kristaiah

2. CONTENTS
• 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 myocardial infraction
 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
 Arteriosclerosis
 Extracorporeal circulation

6.  Anticoagulant
 Haemodialysis
 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. Collect the samplePrinciple
Potency of heparin
(test)determined by
comparing the clotting time
with that of standard.
Requirements
1. Sheep/goat/human
plasma
2. Standard
3. Test
4. Calcium chloride
BIO ASSAY OF HEPARIN
Sodium citrate
Check the quality
0.2%of 1%w/v of CaCl2(clot
within 5min)

8. Bio assay of heparin (flow chart)
Monitered by clotting assays such as the
activated partial thromboplastin time(APIT)

9. Reagents
required to
perform
bioassay
of heparin
Heparin to be
tested
(synthesized)
Standard
preparation of
heparin
solution
Prepared
Plasma
Calcium
chloride

10. 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.

11. Standard heparin
 purified freeze-dried (Pig,Buffalo blood sample)-heparin sodium
salt
•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

12. 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.

13.  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.
 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.

14. Results
S1=T1
• 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.
S1≠T1
• 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.

15. Calculations
• 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'
M= M' + log R
Potency (p)=antilog M= antilog M' ×R

16. 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

17. 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.