Aim : Preparation of Anticoagulated Bulbs

Introduction:
When blood is collected, it clots after sometime. Anticoagulants are the chemicals which prevent clotting
of blood when mixed with blood in proper proportion.

Principle:
The thromboplastin released by damaged tissue, or platelets converts inactive prothrombin into active
thrombin in the presence of calcium ions. Thrombin converts soluble fibrinogen into insoluble fibrin clot
in the presence of calcium ions.

Thromboplastin

Prothrombin Thrombin
Ca++

Thrombin

Fibrinogen Fibrin
Ca++
(soluble) (insoluble)

Fibrin + blood cells clot (fine threads)

Some anticoagulants such as potassium oxalate prevent clotting of blood by precipitating ionic calcium in
plasma, while anticoagulants such as sodium citrate convert calcium ions to unionized form. Since calcium
ions are not available, blood is prevented from clotting. Heparin, however, acts as antithrombin and thus
prevents the formation of thrombin and blood clotting.

Procedure:
• Double Oxalates: 3 parts of ammonium oxalate and 2 parts of potassium oxalate are combined together
to balance swelling effect of ammonium oxalate and shrinking effect of potassium oxalate on the red
blood cells.

Ammonium Oxalate 2.4 g

Potassium oxalate 1.6 g

Distilled water to 100ml

0.2 ml of this solution contains 8 mg of the chemicals which prevent clotting of 3 to 4 ml of blood. 0.2 ml
of this anti coagulated solution is added in each one of the bottles for blood collection. The bottles are
heated in an incubator (or hot air oven) at 60 to 80 C for about one hour. The bottles are taken out of
incubator (or hot air oven) when white layer of anticoagulant appears at the bottom of bottles.
Action: Oxalates combine with calcium in blood to form insoluble precipitate of calcium oxalate
Tests performed by using double oxalates are-
1. Hemoglobin
2. White blood cell (WBC) count
3. Red blood cell (RBC) count
4. Erythrocyte Sedimentation Rate (ESR) determination
5. Packed cell volume (PCV) determination

Disadvantages:
1. WBC morphology is not preserved well, hence it is not generally used for blood smears
2. It is toxic and since calcium oxalate precipitate may cause harm, it is never used for blood banking
purposes and for blood transfusion.

• EDTA(Ethylenediamine tetra acetic acid, disodium or dipotassium salt): 4g/dl of this solution is used to
prepare EDTA bulbs. 0.2ml of this solution is dried per bottle which contains 8 mg of the dry chemical
and prevents coagulation of 3 to 4ml of blood.

Action: It acts as a powerful calcium chelating agent. The calcium in blood is bound in an unionized and
soluble complex with EDTA.

Tests performed using EDTA are-

1. Hemoglobin
2. WBC count
3. RBC count
4. PCV determination
5. ESR determination
6. Platelet count
7. Differential WBC count

Advantages:
1. It gives the best preservation of cellular morphology of the cells is observed even after 2 to 3 hrs of
blood collection.
2. Since platelet clumping is inhibited, for platelet counts using this anticoagulant is preferred.

• Sodium Citrate:
Prepare a 0.106 M solution of trisodium citrate in distilled water:
This is equivalent to 2.73 g/100ml solution of the trisodium salt. Sterilize it. This anticoagulant is used in
solution form and is not dried inside the container. The bottles are marked for 2ml and 10ml. Add 0.4ml
and 1ml of the sterilized anticoagulant solution in bottles marked 2 and 10ml, respectively. After the mixing
of blood specimen with anticoagulant , the ratio of anticoagulant solution and whole blood comes to 1:4
and 1:9 respectively.

Action:It is a calcium chelating agent. It binds with calcium and forms a soluble salt of calcium citrate.

Tests performed using Sodium Citrate :

1. Prothrombin Time (PT)
2. Erythrocyte Sedimentation Rate (ESR)
Disadvantages:
1. It does not preserve the cell morphology