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  • Thromboplastin released by damaged tissue or platelets converts inactive prothrombin into active thrombin in the presence of calcium. Thrombin converts soluble fibrinogen into insoluble fibrin clot in the presence of calcium.
  • Anticoagulant

    1. 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. 2. Coagulation(clotting) mechanism Thromboplastin Calcium
    3. 3. 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.
    4. 4. 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
    5. 5. 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)
    6. 6. 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.
    7. 7. 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.
    8. 8. 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.
    9. 9. 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.
    10. 10. 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.
    11. 11. 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.
    12. 12. Anticoagulant used in Blood Bank ACD (Acid Citrate Dextrose) CPD ( Citrate Phosphate Dextrose ) CPDA (Citrate Phosphate Dextrose Adenine)