Understanding Anticoagulants
The anticoagulant prevents the clotting of blood. It is used in medical laboratories where complete blood or plasma is required, depending upon the test to be done, the type of anticoagulant is decided. Most anticoagulants prevent clotting by removing calcium or iron which are necessary for the clotting process. Every anticoagulant is added in a fixed amount to blood.
Anticoagulants are of the following types:
A. Chemical Anticoagulants
These anticoagulants are prepared in the laboratory. These are as follows.
- Citrate
- Trisodium citrate
- Disodium citrate
- EDTA – Ethylene diamine tetraacetic acid
- CPD – Citrate phosphate dextrose
- Oxalate
- Sodium Fluoride
(i) Citrate:-
Trisodium citrate –
This anticoagulant is prepared by dissolving 3.8 g of trisodium citrate in 100 ml of distilled water. 0.4 ml anticoagulant is required for 2.0 ml of blood. The addition of dextrose to an anticoagulant provides nutrition to the red cells and helps in longer storage. Such anticoagulant is known as Acid Citrate Dextrose (ACD) because along with citric acid, trisodium citrate and dextrose are mixed in this anticoagulant. This anticoagulant is used in the solution form, as it is in the blood bank. The storage of blood with this anticoagulant is a maximum of up to 21 days.
2. Disodium citrate-
This provides the buffering action along with nourishment provided by dextrose. This is used in solution form. The maximum period of storage with this anticoagulant is 21 days.
3.2 EDTA (Ethylene Diamine Tetraacetic Acid)
It can be prepared by dissolving 10 gm of EDTA in 1000 ml of distilled water. 0.4 ml of anticoagulant is required for 2 ml of blood. This anticoagulant does not disturb the cellular structure. Therefore, it can be used for blood cell count, ESR, estimation, etc.
EDTA however, cannot be used for biochemical tests. This anticoagulant is dried in the container by keeping it in an incubator or hot air oven for an overnight period, i.e. for 12 hrs. The storage period with EDTA is 2 to 3 days.
EDTA Advantage
- It is a powerful calcium-chelating agent.
- Used in concentrations of 1.25 to1.75 mg (anhydrous) per ml of blood (i.e. 4 mmol per ml of blood)
- Dipotassium salt is preferred over disodium salt.
- Blood collected in EDTA can be used for TLC, PS preparation, Hb, and DC.
Disadvantage
- Excess of EDTA causes shrinkage of WBCs and RBCs and induces degenerative changes.
- It is unsuitable for coagulation studies.
- EDTA blood fails to demonstrate basophilic stippling of RBCs in lead poisoning.
- Causes leukoagglutination affecting both neutrophils and lymphocytes.
- Activates naturally occurring antiplatelet auto-antibodies which cause platelet adherence to neutrophils.
CPD (Citrate Dextrose)
In this anticoagulant, the citrate is dissolved in phosphate buffer, which maintains pH more accurately than the ACD solution. The dextrose present in this provides nutrition to other cells. The storage period of CPD is 21 days. The disadvantage is that it is costly and difficult to prepare and adjust pH.
Oxalate
It is prepared by dissolving 1.2 gm of ammonium oxalate and 0.8 ml of potassium oxalate in 100 ml of distilled water. 0.2 ml of anticoagulant is required for 2 ml of blood. This anticoagulant may disturb cellular structure if kept for a longer period, however, if used immediately, it can be used for the estimation of bilirubin, and prothrombin time and estimation of blood cells, PVC (Packed cell volume ). This anticoagulant is dried in a container by keeping it for an overnight period in a hot air oven. It is also called double oxalate.
Sodium Fluoride
- It complexes with calcium to form calcium fluoride.
- 30 mg is used for 5 ml of blood.
- It is useful in the estimation of blood glucose levels (Fluoride prevents glycolysis by blocking phosphorylase enzymes in RBCs).
Biological (Natural) Anticoagulant –
Heparin
Heparin is the only biological anticoagulant that cannot be prepared in a laboratory. It is obtained from leech. It is a good anticoagulant and does not alter the size of RBC. It is used in a concentration of 10-15 units/ml of blood. This anticoagulant acts by destroying thrombin or thromboplastin required for clotting.
Heparin is used to determine the blood gases. It can be used used for ESR, PVC, osmotic fragility, and other hematological tests.
Note:-
The anticoagulant should be sterilized before use, and then blood is added to the anticoagulant, it should be mixed gently by inverting it 10 to 15 times or shaking it gently.