Anticoagulants

Anticoagulants used in Hematology & Blood banking

1. ANTICOAGULANTS HAEMATOLOGY & BLOOD BANKING

2. COAGULATION PATHWAY

3. Basic steps of coagulation process Activation of tissue factors(vascular response): Surface activation Thromboplastin activation Thrombokinase production 2.Conversion of prothrombin to thrombin in the presence of thrombokinase and calcium prothrombin Thrombin Thrombokinase,ca ++ 3. Conversion of fibrinogen to fibrin due to the action of thrombin fibrinogen fibrin thrombin

4. ANTICOAGULANT DEFINATION: A substance that prevents blood from clotting by suppressing the synthesis or function of various clotting factors. E.g. it stops blood from clotting. These drugs tend to prevent new clot from forming or an existing clot from enlarging. They don’t dissolve a blood clot.

5. Use: Anticoagulants have various uses- some are used for the prophylaxis or the treatment of thromboembolic disorders. Thrombi are clots. Emboli are clot that break free, travel through blood stream & lodge therein . Used for prevention of vein thrombosis, pulmonary embolism, myocardial infarction & strokes.

6. Ideal characteristics of Anticoagulants: An anticoagulant selected for use in hematological examination must have the following qualities: It must not alter the size of the cell It must not cause hemolysis It must minimize platelet aggregation It must minimize disruption of staining and morphology of leukocytes It must be readily soluble in water It should be soluble in blood It must be keep the blood in fluid condition It should efficiently prevent clotting of blood with minimum quantity.

7. ANTICOAGULANTS USED IN HAEMATOLOGY EDTA HEPARIN SODIUM CITRATE SODIUM FLUORIDE DOUBLE OXALATE

8. Anticoagulants

9. EDTA(ETHYLENE DIAMINE TETRA ACETIC ACID) EDTA is used as a disodium or dipotassium salt.it is also used as a trisodium salt. Disodium and dipotassium salts are solids and trisodium salt is in liquid form. Mostly potassium EDTA is used as an anticoagulant, recommended for hematology studies. EDTA is most frequently used to carry out hematological test, because it preserves the cellular components well. It is also known as sequestrene or vercene . This is a chelating agent that binds the calcium which is needed for coagulation.

10. Mode of action: EDTA binds to metal via 4 carboxylate and 2 amino groups. EDTA forms especially strong complexes with metals. EDTA strongly and irreversibly binds to calcium so act as a powerful calcium chelating agent. Calcium in blood is bound in an unionized and soluble complex with EDTA.

11. MODE OF ACTION OF EDTA

12. CONCENTRATION: It is effective at a final concentration of 1 to 2 mg / mL of blood . It will preserve blood excellently for at least 6 hrs. Refrigeration will extend the preservation to 24 hrs.

13. Disadvantage: Excess of EDTA affects both red blood cells & leucocytes causing shrinkage & degarerative changes. Excess of EDTA may cause decrease in packed cell volume (PCV) & increase in mean cell Hb concentration (MCHC) Platelet swell & disintegrate due to excess of EDTA & artificially high platelet count may be obtained due to disintegrated platelets. EDTA interferes with blood chemistry tests as follows: 1. False decrease in alkaline phosphatase by binding Mg+ ². 2. Decrease the CO 2 combining power of blood. 3. Interferes with Jaff reaction for creatinine test. 4. Alters Na+, K+, Ca+ concentration in plasma. 5. EDTA affects the function of fibrinogen ( can sometimes be seen as stranding of fibrin on a blood smear)

14. Advantages: It gives best preservation of cellular morphology after 2 to 3 hrs of blood. It inhibits platelet clumping so useful in platelet count. Other uses: Food preservative, textile industry, pulp & paper industry, oil production etc.

15. B. Heparine It is a natural anticoagulant in the body, found in the liver, and may also be with in basophils and mast cells, heparin also called anti thromboplastin or antithrombin. It is available in a liquid or dry form as…… sodium, calcium, ammonium and lithium salt, Each of these will interfere with determination of their respective ions in the plasma 

16. It was first discovered by JaymMclean in 1916 & William henry Howell in 1918. It is known as anti-thromboplastin or anti-thrombin Mode of action: It interfers in the formation &/ or activity of thrombin & activity of clotting factor X, XI, XII, IX By this action it stops formation of thrombin from prothrombin & thus stopping the formation of fibrin from fibrinogen.

17. Mode of action

18. Concentration: the optimum conc. is 0.1- 0.2 mg/ml of blood. Advantage: Heparin is the choice of Anticoagulant for blood pH,and blood gas Analysis. Acid base balance. It may be used for special trace elements studies and some cytology . Excessive heparin does not alter the RBC volume It is used in vivo in suspected thrombosis & pulmonary embolic condition to prevent intravascular coagulation. It is also used for enzyme studies. It is used for osmotic fragility testing. Used for cell culture

19. Disadvantages : It causes clumping of leukocytes It interferes with staining of leukocytes. It is the most expensive of the anticoagulant Blood clot in 8-12 hrs because clotting is only delayed and not prevented. It is not suitable for agglutination tests , and coagulation studies It may interfere with some automated biochemical analysis of plasma.

20. 3. Sodium Citrate A citrate can refer to the conjugate base of citric acid to esters of citric acid. Molecular formula of C 6 H 5 Na 3 O 7 (sodium citrate). An example of former, a salt is trisodium citrate an ester is trimethyl citrate. It is used in liquid form.

21. Mode of action: It removes calcium ions by forming soluble calcium citrate complex. So it converts ionised calcium into unionised soluble complex.

22. Advantages: In the ESR determination by westergen method. (2 ml marked) test tube 0.4ml of 3.8 g/dl trisodium citrate solution is taken & blood is added upto mark (1.6 ml of blood). Nowadays EDTA is used instead of citrate. In Prothrombin time determination. In a (2.5 ml marked) test tube 0.25ml of 3.8 g/dL sodium citrate dilution is taken & blood is added upto mark 2.25ml of blood) for this test 3g/dl sodium citrate is required.

23. Disadvantages It interferes with many chemical tests Used alone it preserves blood for only few min. It has a tendency to shrink cells. Because of 10% dilution of blood – sodium citrate is generally not used for CBC It inhibits enzyme activities such as SGPT, SGOT & alkaline phosphatase & interfers in the determination of calcium & inorganic phosphorus

24. Uses: It is used in liquid form & it forms calcium citrate complex which is soluble so it is also used for coagulation studies. ESR by westergen method Reticulocytes count Act as a both diluent and anticoagulant Heinz body detection Trisodium citrate is employed as a flavouring agent.

25. 4. Sodium Fluoride & Potassium Oxalate These tubes contain mixture of fluoride & oxalates. Sodium fluoride is a weak anticoagulant therefore it is used with potassium oxalate. Mode of action: Sodium fluoride inhibits the glycolytic enzyme responsible for the breakdown of glucose in blood. Blood glucose concentration decrease by about 10 mg/dl per hr at 25 C if specimen is untreated.

26. Mode of action

27. Uses: It is used for blood glucose determination Blood urea estimation Disadvantages: Fluoride plasma is not suitable for enzyme determination because it inhibits enzyme activity.

28. 5. Double Oxalate Types : A. Potassium oxalate B. Ammonium oxalate Three parts of Ammonium oxalate and two parts of Potassium oxalate are combined together & forms Double Oxalate. This two oxalates are used in combination, because they balance the adverse effect of each other that is swelling effect of ammonium oxalate & shrinking effect of potassium oxalate on red blood cells.

29. The solution of double oxalate is prepared as: 0.2 ml of this solution contains 8 mg of the chemical which prevents clotting of about 3 to 4 ml of blood. Ammonium oxalate 2.4 g Potassium oxalate 1.6 g D/w 100 ml

30. Mode of action: Oxalate combine with calcium in blood to form insoluble precipitate of calcium oxalate. By this action it inhibits the coagulation of blood. These acts by chelating calcium . Calcium oxalate is formed as insoluble precipitate, these are used for blood chemistry and hematocrit.

31. Use Double oxalate is used for Hb studies, RBC count & WBC count. ESR rate determination by wintrobe’s method It is also used for packed cell volume (PCV) determination. Disadvantages: WBC morphology is not preserved well hence it is not generally used for blood smear.it may cause harm, it is never used for blood banking for blood transfusion.

32. Anticoagulant use in Blood Banking

33. Introduction Blood has to be drawn in proportion with the amount of anticoagulant in the blood bag The ratio of anticoagulant preservation solution to blood collected should be 1.4: 10ml The different anticoagulant solutions are added to blood to preserve blood, to prevent clotting, to preserve functions of various cellular components & to prevent denaturation of plasma proteins. Blood bags are specially designed to collect the following volumes: 350ml of blood bags: in which 350ml of blood is collected into 49 ml of anticoagulant solution 450ml of blood bags: : in which 450ml of blood is collected into 69 ml of anticoagulant solution The first anticoagulant preservative was introduced by Rous & Turner in 1916. It consist citrate and glucose solution. In 1943 during 2 nd world war, an acidified citrate dextrose solution was introduced by loutit and Mollison. It is used for preservation of blood. The different anticoagulant preservative solution available for whole blood collection & storage are as follows:

34. Anticoagulant use in Blood Banking Acid Citrate Dextrose (ACD) Citrate Phosphate Dextrose (CPD) Citrate Phosphate Dextrose Adenine (CPDA)

35. Acid Citrate Dextrose (ACD) Blood stored in ACD solution at 2-6°C for 21 days. It has more than 70% erythrocyte survival at 24 hrs post transfusion. It contains: Tri sodium citrate (dihydrate) 22.0 g Citric acid (monohydrate) 8.0 g Dextrose 24.6 g D/W made to 1 L pH 5.0-5.1

36. Citric acid prevents caramelization of glucose in citrate dextrose solution during autoclave. Citric acid is a weak acid and along with citrate it gives optimal pH which has a least deleterious effect on red cells. Citrate act as an anticoagulant and prevent clotting by chelating calcium. It inhibits calcium dependent step of coagulation pathway. Dextrose: It is necessary for metabolism of stored red cells. Sufficient dextrose is needed for ATP generation via glycolytic pathways enhanced ATP levels in red cells indicates enhanced post transfusion viability.

37. Citrate Phosphate Dextrose (CPD) In 1957 Cibson showed that by adding phosphate to acid citrate dextrose solution post-transfusion survival rate of red cells could be increased. It contains: Tri sodium citrate (dihydrate) 26.30 g Citric acid (monohydrate) 3.27 g Sodium dihydrogen phosphate(mono) 2.22 g Dextrose 25.50 g D/W made to 1 L

38. Sodium dihydrogen phosphate act as a buffer to control decrease in pH expected from build up of lactic acid, an end product of glycolysis. The blood stored in CPD has better post transfusion survival after 21 days than ACD due to slightly higher pH because of phosphate compound.

39. CPDA-1 Simon in 1962 showed that in modified CPD solution supplemented with adenine the 24 hrs post transfusion survival of red cells was 80.5 ± 6.5% after 35 days storage. CPDA-1 is anticoagulant preservative used in recent blood banks.

40. It contains, Adenine provide a substrate for synthesis of ATP in red cells. Which result in increased viability of red cells. Blood storage in CPDA-1 has improved the post transfusion survival of red cells to 35 days because of enhanced ATP production and better preservation of 2,3 DPG (2,3 Diphospho -Glycerate) Tri sodium citrate (dihydrate) 26.30 g Citric acid (monohydrate) 3.27 g Sodium dihydrogen phosphate(mono) 2.22 g Dextrose 31.08g Adenine 0.275 g D/W made to 1 L

41. CPDA-2 CPDA-2 has all constituent same as CPDA-1. The difference between these two is conc. of dextrose and adenine is different. In CPDA-2 the amount of dextrose is increased to 44.6 g And amount of adenine to 0.55 g. It is better than CPDA-1 due to more dextrose and adenine contents.

42. Anticoagulants use in Blood bank