1. Anticoagulant
When Blood is collected, it clots after sometime. The
anticoagulants are the chemical agents which prevent the
clotting of Blood when mixed with Blood in proper
proportion.
Purpose of using anticoagulants:
- For study of various constituents of Blood components.
- Study of coagulation(clotting of Blood).
- Preservation of Blood in Blood Bank.
Properties of anticoagulant
-
It must be soluble in Blood.
It must be keep the blood in fluid condition,
It must not be bring haemolysis of Blood cells.
It must not be change the size of RBC.
It must minimize destruction of leukocytes.
It must minimize aggregation of platelets.

2. Coagulation(clotting) mechanism
Thromboplastin
Calcium

4. Classification of
anticoagulant
Bind with Calcium
2. Non-calcium chelator
Do not bind with Calcium
 Oxalates
 Heparin
It is following forms:-Ammonium Oxalate
-Potassium Oxalate
- Double Oxalate
 Warfarin etc.
1.Calcium chelator
 EDTA (Ethylenediaminetetraacetiacid)
 Tri-sodium citrate etc.
Most of the anticoagulants used in laboratory act by binding with calcium and
prevent clotting of blood since calcium ion is essential for many of the steps in
coagulation mechanism.

5. Oxalates
Ammonium oxalate:This is used at a concentration of 2 mg for 1 ml of Blood.
This anticoagulant causes swelling of the RBC therefore, it is not
recommended for use with blood for PCV,ESR and cell morphology.
Potassium oxalate:It is used at a concentration of 2 mg for 1 ml of Blood. This
anticoagulant is most often used for chemical analysis. It causes
shrinkage of RBC therefore it is also is not recommended for the study
of PCV,ESR and other cell morphology.
Double oxalate:Ammonium oxalate and Potassium oxalate are combined together
to balance the swelling effect of Ammonium oxalate and the shrinking
effect of Potassium oxalate on the red cells. It is the mixture of 3 parts
of Ammonium oxalate and 2 parts of Potassium oxalate which is
prepared as follows:o Ammonium oxalate
2.4 gram
o Potassium oxalate
1.6 gram
o Distilled water
100 ml

6. E.D.T.A.(Ethylenediamine tetra-acetic acid)
It is the most commonly used anticoagulant in
Haematology lab because E.D.T.A. is the most
powerful calcium chelating (binding) agent we have. It
gives the best preservation to the cell morphology
therefore E.D.T.A. is the preferred anticoagulant for
all cell count and blood smear preparation.
E.D.T.A. is used in two different forms:Di-sodium E.D.T.A. salt (Versene)
Di-potassium E.D.T.A. salt (Sequestrene)

8. Preparation of E.D.T.A vials (bulb) in your laboratory
4 gm/dl of this solution is used to prepare EDTA bulbs.
20µl of this solution is dried per bottle which contains 0.8 mg of
dry chemical and prevents coagulation of 1 to 2 ml blood.
Required materials
1. Physical balance
2. EDTA powder
3. Distilled water
4. Conical flask
5. Measuring cylinder
6. Micropipette (20µl capacity)
7. Spatula
8. Hot air oven
9. Vials
10.Storing bottle etc.

9. e
Keep all the required materials in the working table.
2. Put the physical balance in plane surface and balanced it properly.
3. Put the equal sizes of paper pieces in both sides of pan and balance it
again.
4. Put 4 gm weight in one side of the pan.
5. Put EDTA powder on the other side of the pan slowly till it shows
equals to the 4 gm weight.
6. Put the EDTA powder in the conical flask
7. Measure100 ml of distilled water by using measuring cylinder, pour
into the conical flask containing EDTA powder and mix well.( Heat
can be applied for proper mixing of the solution).
8. Pipette 20µl of anticoagulant solution in each clear and dry vials.
9. Put the vials containing anticoagulant solution in the hot air oven at
the temperature of 60oC for 30 minutes.
10. After drying EDTA powder remain attached at the bottom of the
vials. Store the EDTA vials by putting lid over it and keep in safe place
which can be used for 1-2 ml of blood sample.
1.

10. Calculation
4 gm% EDTA solution
in 100 ml of distilled water (D.W.)= 4 gm of EDTA
in (100 x 1000)µl of D.W.= (4 x 1000) mg of EDTA
4000
1 ,,
,,
=
——
mg
100000
4
20 µl
,,
,,
=
—— x 20 mg
100
= 0.8 mg of EDTA.

11. Excess of
EDTA
Excess of EDTA affects both red blood cells
and leukocytes causing shrinkage and
degenerative changes.
Excess of EDTA ( in case of 2 mg/ml) may
cause significant decrease in packed cell
volume(PCV) and increase in mean cell
haemoglobin concentration (MCHC).
Platelets swell and disintegrate due to the
excess of EDTA and artificially high platelets
count may be obtained due to disintegrated
platelets.

12. Tri-sodium citrate
It is used as a liquid form.
It binds with calcium.
The concentration of Tri- sodium citrate is used as 3.8%.
For PT (Prothrombin Time) a kind of coagulation test,
3.8% Tri-Sodium citrate is used at the ratio of 1:9 i.e. 1
part of anticoagulant and 9 part of Blood.( 200 µl of 3.8%
Tri-Sodium citrate and 1.8 ml of Blood is used.)
Fro
ESR (Erythrocyte Sedimentation Rate) by
Westerngreen method
1 part of 3.8% Tri-Sodium citrate is mixed with 4 part of
Blood that is the ratio of 1:4.( 400 µl of 3.8% Tri-Sodium
citrate and 1.6 ml of Blood is mixed).
It do not preserve the cell morphology.

13. Heparin
It is a natural anticoagulant and is
normally present in the blood in small
amount and highly acidic. This is the best
anticoagulant for open heart surgery and
it causes minimum haemolysis.
It is very expensive.
It produce black back ground in the
smear so it is not use for smear
preparation.

14. Anticoagulant used in Blood Bank
ACD (Acid Citrate Dextrose)
CPD ( Citrate Phosphate Dextrose )
CPDA (Citrate Phosphate Dextrose Adenine)