1. BY Dr. Richa Sharma MODERATOR: Dr. Shariq Ahmad ANTI COAGULANTS

2. History Physiologist Johannes Mller described fibrin, the substance forming thrombus serving as the first milestone for anti coagulants. Arthus discovered in 1890 that calcium was essential in coagulation. The theory that thrombin is generated by the presence of tissue factor was consolidated by Paul Morawitz in 1905. First anti coagulant preservative was used by Rous & Turner in 1916 contained of citrate-glucose solution and was used to store human blood during first world war in 1987. Heparin is one of the oldest drugs currently in widespread clinical use. It was originally isolated from canine liver cells, hence its name Heparin. Heparin's discovery can be attributed to the research activities of two men: Jay McLean and William Henry Howell. 1950s Mark Rubin at Georgetown University 3. DEFINITION A substance that prevents coagulation or clotting of blood but doesnt dissolve an already formed clot. Uses Storage of blood for blood transfusion or hematological testing Therapeutic 4. Number and/or name Function I (fibrinogen) Forms clot (fibrin) II (prothrombin) Its active form (IIa) activates I, V, VII, VIII, XI, XIII, protein C, platelets Tissue factor(formerly known as factor III) Co-factor of VIIa Calcium(formerly known as factor IV) Required for coagulation factors to bind to phospholipid V (proaccelerin, labile factor) Co-factor of X with which it forms the prothrombinase complex VI Unassigned old name of Factor Va 5. VII (stable factor, proconvertin) Activates IX, X VIII (Antihemophilic factor A) Co-factor of IX with which it forms the tenase complex IX (Antihemophilic factor B or Christmas factor) Activates X: forms tenase complex with factor VIII X (Stuart-Prower factor) Activates II: forms prothrombinase complex with factor V XI (plasma thromboplastin antecedent) Activates IX XII (Hageman factor) Activates factor XI, VII and prekallikrein XIII (fibrin-stabilizing factor) Crosslinks fibrin 6. 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 7. COLOUR CODING OF VIALS 8. EDTA FOR ROUTIN E HAEMAT OLOGY BUFFER ED SODIUM CITRATE FOR COAGUL ATION STUDIES POTTASI UM OXALTE OR SODIUM FLOURID E GLUCOS E DETERM INATION NO ADDITIV E COLLEC TION OF SERUM ACD PRESE RVE RBC FOR BLOOD BANKI NG & HLA Typing HEPARIN INHIBIT THROMBI N ACTIVATI ON 9. ETHYLENE DIAMINE TETRA ACETIC ACID Used for routine hematological work MOA: EDTA has chelating effect on calcium molecule in blood. Three types of EDTA salt sodium potassium lithium Dipotassium salt more soluble than Disodium salt, hence preferred Although Dilithium salt is less soluble than Dipotassium but the same sample of blood can be used for chemical investigations 10. Lavender cap Anticoagulant conc: EDTA-K2 1.5-2.0mg/ml 11. The salt can be used in powder as well as liquid form Coating of inside surface of blood collection tube with thin layer of EDTA improve speed of its uptake by blood Tripotassium salt dispensed in liquid form in blood will be slightly diluted and produces some shrinkage in RBC resulting in 2-3% dec. in PCV with in 4hrs of collection followed gradual inc. in MC V Trisodium EDTA not recommended d/t high pH K2EDTA and Na2EDTA salts are commonly used in dry form; K3EDTA is normally used in liquid. K2EDTA is spray-dried on the wall of the tube and will not dilute the sample and is recommended by ICSH and CLSI for haematology testing. 12. USES OF EDTA TLC DLC ESR (by wintrobe method) CBC RETICULOCYTE COUNT PLATELET COUNT LEAD POISONING G6PD DEFICIENCY 13. DISADVANTAGES OF EDTA RBC & WBC shrinkage degenerative changes >2mg/ml blood : dec. PCV ; inc. MCHC PLATELETS swell & disintegrate artificially high platelet count OTHERS Causes leucoagglutination affecting both lymphocytes & neutrophils also there is flowering of lymphocytes which can be counted as neutrophil. Fails to demonstrate basophilic stippling of RBC in lead poisoning. Monocyte activation measured by release of tissue 14. DOUBLE OXALATE Acts by chelating calcium in blood Also k/a Ammonium & Potassium oxalate OR Heller and Paul double oxalate Ammonium salt : causes swelling of RBC Potassium salt : causes cell shrinkage hence double oxalate retains normal shape of RBC proportion of potassium : ammonium :: 2:3 15. USES ESR Routine Hematology (TLC, DLC, Hb) DISADVANTAGES Variable dilution of plasma is caused by potassium oxalate d/t water transport from cells to plasma. If oxalate is added to vials and dried in an oven, great care is to be taken to avoid temperatures above 80C. As Oxalates are converted to carbonates by prolonged exposure to elevated temperatures. 16. TRISODIUM CITRATE Acts by chelating calcium in blood . It is used in a concentration of :- 1 part 0.11 M sodium citrate to 9 parts whole blood for ESR (by westergren) 1 part 0.11 M sodium citrate to 4parts whole blood for coagulation profile. Citrate is usually in blue Vacutainer tube. It is in liquid form in the tube and is used for 17. USES FIBRINOGEN PT PTT ESR(by westergren method) DISADVANTAGES Alters concentration of blood as it is always used in solution form , hence, is not used for routine hematology. 18. SODIUM FLOURIDE Sodium fluoride has a double action on the blood: It prevents clotting by chelating calcium. It prevents all phosphatase action, inhibit glucose oxidase activity in enzymatic glucose reaction. It is used for determination of blood sugar. It preserve blood for 24 hours at room temperature and 4 to 6 day in refrigerator. 19. Common disadvantages with calcium chelators They inhibit various plasma enzyme activities like: Amylase activity inhibited by oxalate and citrate LDH and Acid Phosphatase inhibited by oxalate Fluoride , Heparin or EDTA interfere with accurate determination of electrolytes 20. HEPARIN Heparin is a naturally-occurring anticoagulant produced by basophils and mast cells. Heparin acts as an anticoagulant, preventing the formation of clots and extension of existing clots within the blood. While heparin does not break down clots that have already formed (unlike tissue plasminogen activator), it allows the body's natural clot lysis Mechanism of action: Heparin binds to anti thrombin inhibiting interaction of various clotting factors i.e. Xa, IX a, XIa, XII a & plasmin. 21. AMOUNT FOR BLOOD STORAGE: 5 10 IU /mL of blood SAMPLE COLLECTION: 0.5- 2.0 IU/mL . 22. USES ESR Methaemoglobin Osmotic Fragility HLA Typing Ammonia Disadvantages Costly In Leishman stained peripheral blood film blue color is imparted to the background due to presence of plasma 23. ANTICOAGULANTS FOR BLOOD STORAGE ACD (Acid Citrate Dextrose) CPD(Citrate Phosphate Dextrose) CPDA-1(Citrate Phosphate Dextrose Adenine) HEPARIN 24. COMPOSITION( in 1l of distilled water) CHEMICAL ACD CPD CPDA-1 TRI SODIUM CITRATE 22.0 g 26.3 g 26.3 g CITRIC ACID 8.0 g 3.27 g 3.27 g DEXTROSE 24.6 g 25.50 g 31.8 g SODIUM DIHYDROGE N PHOSPHATE ____ 2.28 g 2.22 g 25. 15ml of ACD and 14ml of CPD & CPDA-1 is required for preservation of 100ml of blood Initial pH of ACD 5.0 and of CPD & CPDA-1 is 5.6 Also storage time at 2-6C for ACD & CPD is 21 days and for CPDA-1 is 35days 26. FUNCTION OF VARIOUS COMPONENTS o CITRATE Causes chelation of calcium SODIUM DIPHOSPHATE Prevents fall in pH o DEXTROSE Addition of glucose prolongs survival of stored RBC as it is required for metabolism. Glucose passes from plasma to RBC and is utilized for energy production. 2 PATHWAYS for energy production : 90% by Embedem Mayeroff pathway in which there is breakdown of glucose into lactate through anaerobic glycolysis. 27. 10% by Pentose phosphate pathway through aerobic glycolysis. The various intermediaries formed are necessary for maintaining their ability to deliver oxygen to tissues through generation of 2,3-DPG. Viability correlates with the level of ATP. o CITRIC ACID Fairly weak tribasic hydroxyacid Along with tri sodium citrate which is alkaline gives an optimal pH. Prevents carmalization of glucose in citrate dextrose solution during autoclaving. o ADENINE 28. ADDITIVE SYSTEM To extend RBC storage to 42 days and to harvest maximum amount of plasma, additive systems are now available in which storage environment of RBC is altered by adding certain nutrients after removal of plasma Its made by adding the following components to the CPD :- Sodium chloride-adjusts osmotic pressure Adenine maintains high level of ATP in RBC Dextrose Mannitol- prevents disintegration of RBC 29. Changes in stored blood 30. PHYSICAL o Shape of RBC discoid to spherical o Loss of lipid in RBC membrane o Increased cellular rigidity d/t decrease in deformability o Decrease in critical hemolytic volume(CHV) in parallel with membrane lipid content CHV is largest volume to which RBC swells before haemolysis 31. o Decrease in osmotic fragility o Blood stored for >24 hr at 2-6C has few viable platelets and granulocytes o Heat labile coagulation factors V & VIII decrease on storage upto 50% in first 48-72hrs There after loss is comparatively slower after 21 day factorV 30% factorVIII 15% 32. BIOCHEMICAL CHANGES Drop in pH and dextrose level causes anaerobic glycolysis in RBC to generate ATP. Decreased ph causes Decreased 2,3-DPG and cells ability to release oxygen to the tissues. Metabolic functions slow down in cold temperature, ATP levels decreases. ELECTROLYTE: loss of potassium from RBC to plasma passage of sodium from plasma to cells plasma ammonia levels also increase 33. THERAPUTIC USES Anti coagulants used for therapeutic use are :- Heparin and LMWH Oral Anticoagulants warfarin and other Vit K antagonists They are used for prevention of various thrombotic diseases DVT PULMONARY EMBOLISM ACUTE MYOCARDIAL INFRACTION 34. THANK YOU