ANTICOAGULANT

DEFINITION A substance that prevents coagulation or clotting of blood but doesn’t dissolve an already formed clot. Uses Storage of blood for blood transfusion or hematological testing Therapeutic

Anticoagulant Purpose of using anticoagulants: 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.

Commonly used anticoagulants EDTA(Ethylene Diamine Tetra Acetic acid) Double oxalate Sodium citrate Sodium Fluoride Heparin ACD(Acid Citrate Dextrose) CPD(Citrate Phosphate Dextrose) CPDA(Citrate Phosphate Dextrose Adenine)

FOR ROUTINE HAEMATOLOGY ACD EDTA FOR ROUTINE HAEMATOLOGY BUFFERED SODIUM CITRATE FOR COAGULATION STUDIES POTTASIUM OXALTE OR SODIUM FLOURIDE GLUCOSE DETERMINATION NO ADDITIVE COLLECTION OF SERUM ACD PRESERVE RBC FOR BLOOD BANKING & HLA Typing HEPARIN INHIBIT THROMBIN ACTIVATION

Non-calcium chealeter Anticoagulant Calcium chealeter Oxalates Ammonium oxalate Potassium oxalate Double Oxalate E.D.T.A. Tri-Sodium citrate Non-calcium chealeter Heparin Warfarin

Classification of anticoagulant 1.Calcium chelator Bind with Calcium Oxalates It is following forms:- -Ammonium Oxalate -Potassium Oxalate - Double Oxalate EDTA (Ethylenediaminetetraacetiacid) Tri-sodium citrate etc. 2. Non-calcium chelator Do not bind with Calcium Heparin Warfarin 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.

CLASSIFICATION USED IN VIVO Parenteral anticoagulants These are drugs used to reduce the coagulability of blood classified into: USED IN VIVO Parenteral anticoagulants a) Heparins (ITI) High Molecular Weight Heparin Unfractionated Heparin (UFH) Low molecular weight heparin (Enoxaparin, Dalteparin, Tinzaparin, Reviparin, Danaparoid) b) Heparinoids (DTI)-Heparan sulfate, Hirudin, Lepirudin, Bivalirudin, Argatroban 1. Indirect Thrombin Inhibitors: HMWH, LMWH 2. Direct Thrombin Inhibitors: Hirudin, Lepirudin, Bivalirudin, Argatroban

Oral anticoagulants (1)Coumarin derivatives :Bishydroxycoumarin (dicumarol), Warfarin sod, Acenocoumarol (Nicoumalone), Ethylbiscoumacetate (2)Indandione derivative: Phenindione. USED IN VITRO (a)Heparin (b)Sod. citrate :used in blood banks to store the blood (c)Sod. oxalate : (d)Sod. editate : used as an anticoagulant in laboratory

Ammonium oxalate:- Potassium oxalate:- Oxalates 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:- Ammonium oxalate 2.4 gram Potassium oxalate 1.6 gram Distilled water 100 ml

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)

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.

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.

Intrinsic Pathway Extrinsic Pathway Vit. K dependent Factors Blood Vessel Injury Tissue Injury Tissue Factor XII XIIa HMW-Kininogen Thromboplastin XI XIa XIIa, Xia, Kallikrein ca+ IX IXa VIIa VII ca+ ca+ X Xa X XIII ca+ ca+ Thrombin Prothrombin Factors affected By Heparin XIIIa Fibrinogen Fribrin insoluble Vit. K dependent Factors Affected by Oral Anticoagulants Stabilised Fibrin threads

Mechanism + Heparin No heparin Active clotting factors Slow AT III Inactive clotting factors Heparin Active clotting factors Fast AT III + Inactive clotting factors

ORAL ANTICOAGULANTS

Vitamin K-Dependent Clotting Factors VII The four Vitamin K dependent clotting factors are synthesized in the liver. IX X II Synthesis of Functional Coagulation Factors

Warfarin Mechanism of Action Vitamin K VII Antagonism of Vitamin K Warfarin acts as an anticoagulant by blocking the ability of Vitamin K to carboxylate the Vitamin K dependent clotting factors, thereby reducing their coagulant activity. IX X II No Synthesis of Functional Coagulation Factors Warfarin

Mechanism of Action Coumarins block the Gamma Carboxylation of glutamic acid residues of Clotting factors II,VII, IX, X , endogenous anti-coagulants C & S. This is coupled with oxidative deactivation of Vit K Coumarins and Indanediones (-) enzyme Vit K epoxide reductase that converts Vit K epoxide to its active hydroquinone (reduced form) Thus they prevent the activation of Vit K and hence along with it carboxylations of clotting factor residues

Why Carboxylation is necessary? Necessary for ability of clotting factors to bind Ca+ and to get bound to phospholipid surfaces which is necessary for coagulation Factor VII affected first, then IX, X, and finally Factor II (depends upon half lives of circulating factors)

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