Presented by Nabiilah & Naailah

Date: 4th Feb 2015

• Introduction on Heparin

– Biological activity of Heparin

– Therapeutical Uses

• Assay of Heparin

– Principle

– Standard Preparations

– Methods

– Results

– Calculations

– Limit of Errors

• Conclusion

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.

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

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

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.

Bioassay of Heparin• The potency of heparin sodium is

determined by comparing theconcentration necessary to prevent theclotting of plasma with theconcentration of the StandardPreparation of heparin sodiumnecessary 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

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

•The potency of standard heparin is determined inrelation to the International Standard stated by theWorld Health Organization.

Test solution:

•Accurately about 25 mg of the test sample is weighed

•Sufficient saline is added to give the concentration of 1mg/ml

Standard heparin: purified freeze-dried heparin sodium salt

Standard heparin:

Method: In clean test-tubes, graded amount of the solution of standardpreparation is added (the largest dose not exceed 0.8 ml)

Sufficient volume of saline is then added to make total volume of 0.8ml and add 1 ml of prepared plasma to each test tube

0.2 ml of 1 % w/v solution of calcium chloride is added, thetime is noted and each tube is immediately stoppered with asuitable stopper

The contents are mixed by inverting three times in such a way that the entire inner surface of the tube is wet

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.

• 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

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S1T1

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Results:

• 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:

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

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.

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