1. Dr : AMER WAHAN

2. Anticoagulants

3. Anticoagulants
• ANTICOAGULANTS
• Blood coagulates when withdrawn from the vessel and anticoagulants
are used to prevent blood from clotting.
• Commonly used anticoagulants are
• I. Calcium chelating agents
• a. EDTA
• b. Double oxalate
• c . Sodium citrate solution
• d. Sodium fluoride
• II. Heparin

4. Calcium Chelating Agents
• Most of the anticoagulants used in the laboratory act by
binding calcium as an insoluble salt or soluble but un-ionized
salt.
• Since calcium is essential for many of the steps in the clotting
mechanism, chelation of calcium prevents clotting.

5. Calcium Chelating Agents
• Calcium chelating agents Anticoagulant Include:
• a. EDTA
• b. Double oxalate
• c . Sodium citrate solution
• d. Sodium fluoride

6. EDTA
(Ethylene Diamine Tetra Acetic acid)
EDTA (Ethylene Diamine Tetra Aceticacid): Commonly disodium or
tripotassium salts areused in the laboratory.
• Action:
They bind and remove free calcium ions from blood by chelation,
thereby preventingcoagulation.
• Advantages:
• it is the anticoagulant of choice for all cell counts and peripheral
smear.
• 1- EDTA preserves the morphology of the blood cells even upto 3
hours
• 2 preserves the cell counts upto 12 hours after collection of the blood.

7. EDTA
(Ethylene Diamine Tetra Aceticacid)
• Tripotassium salts of EDTA are more soluble and arepreferred for cell
counters than that of less soluble sodium salt.
• EDTA is used in solid form at concentration of 2 mg/mL of blood.
• Disadvantages:
• It is not suitable for coagulation studies

8. EDTA
(Ethylene Diamine Tetra Aceticacid)
• Alterations induced by EDTA:
• RBC: Red cells may assume spherocytic or macrocytic form.
• WBC: Morphologic changes take place in the neutrophils within 30 minutes of
blood collection.
• Platelets: They swell and disintegrate and a false decrease in their count may
be observed.
• Uses
• Cell counts: RBC count, total leukocyte count,, platelet count.
• peripheral smear
• Hemoglobin, HbF estimation and Hb electrophoresis.

9. b. Oxalate
• Potassium oxalate:
Most often used for chemical analysis.
• Shrinks RBCs and therefore notrecommended for packed cell volume (PCV),
erythrocyte sedimentation rate (ESR), orperipheral smear.
• Ammonium oxalate:
It causes swelling of RBCs and therefore not used for PCV, ESR or peripheral
smear.

10. Oxalate
• Double oxalate:
To balance the swelling effect of ammonium oxalate and the
shrinking effect of potassium oxalate,
the two are combined in a mixture in the ratio of 3 parts of
ammonium oxalate to 2 parts of potassium oxalate.
• Morphology of the blood cells is not well preserved; hence it has
now been replaced by EDTA.
• As it is toxic, it is not used as an anticoagulant for blood
transfusion.
• It is used in concentration of 2 mg/mL of blood.

11. Sodium Citrate
• Action:
• Sodium citrate binds with calcium ions in the blood to form a
soluble calcium citrate complex thus preventing coagulation of
blood.
• Disadvantages:
• Since it is used as a liquid which results in dilution of cellular
elements, it is not suitable for cell counts, Hb, etc.

12. Sodium Citrate
• Uses
1. Coagulation studies:
Either 3.8% trisodium citrate pentahydrate or 3.2% trisodium citrate
dihydrate solution is used for coagulation studies. One part of anticoagulant
is mixed with 9 parts of blood (1:9, i.e. ratio of 0.5 mL: 4.5 mL.)
2. ESR by Westergren method:
In the Westergren method of ESR estimation, ratio of 1:4 between citrate and
blood.

13. Sodium Citrate
1. Blood Bank:
• ACD Solution (acid citrate dextrose solution) was used as an anticoagulant in
blood banking. This has been replaced by citrate phosphate dextrose adenine
(CPDA).

14. Sodium Fluoride
• Sodium Fluoride
Sodium fluoride acts an antiglycolytic agent which inhibits the use of
glucose by blood cells.
• Use:
It is used for estimation of blood sugar.

15. Heparin
• Action:
Heparin acts as an anticoagulant by preventing conversion
of prothrombin to thrombin
• Method
Syringe is heparinised by taking 1 drop of heparin to wet the
inner surface of the syringe and the blood is collected.

16. Heparin
• Disadvantages
1. Expensive
2. Causes clumping of platelets and leukocytes, so it is not used for
automated cell counters.
3. Imparts bluish discoloration to the background of peripheral blood
smears.
4. Prevents coagulation for only a limited period of time and its effect
slowly disappears as the heparin is metabolized or neutralized in the
blood.

17. Heparin
•Uses
1. Osmotic fragility test done for diagnosis of
spherocytosis.
2. Red cell enzyme studies like G6PD and PK
deficiency.
3. Electrolyte estimation.
4. Arterial blood gas analysis.