BLOOD PRESERVATIVES

Blood preservatives

1. BLOOD PRESERVATIVES Dr. ZIKRULLAH

2. The process by which blood or its components are kept viable outside the body (i.e., kept away from decay by means of a chemical agent, cooling, or a fluid substitute that mimics the natural state within the body) is called blood preservation. Goal of blood preservation - to provide viable & functional blood components for patients requiring blood transfusion. DEFINITION

3. PHYSICAL Disc changes Changes in deformability due to loss in membrane lipids Increased osmotic fragility Loss of heat labile coagulation factors Apoptotic changes BIOCHEMICAL Decrease in ATP levels Build up of lactic acid Decrease in pH Decrease in 2,3 DPG levels leading to shift of oxygen dissociation curve Clotting of blood STORAGE CHANGES

4. When blood is stored at 2-6 °C, glycolysis is reduced but does not stop. Glycolysis results in the production of lactate, with subsequent decrease in pH , which alters red cells viability. Preservative solutions provide buffering capability to minimize pH changes and optimize the storage period. DECREASE IN pH

5. A fall in pH in the stored blood results in – decrease in red cell 2,3-DPG level by inhibiting RBC glycolysis which results in increase in hemoglobin-oxygen affinity. DPG-depleted red cells have impaired capacity to deliver oxygen to the tissues. DECREASE IN 2,3 – DPG LEVEL

6. However , after transfusion the red cells continue to synthesize 2,3-DPG and levels return to expected normal values within 24 hours. Rate of restoration of 2,3-BPG is affected by acid-base status of recipient phosphorous metabolism degree of metabolism

7. Decrease in ATP results in Leak of Na and K ion from the cell membrane Increased osmotic fragility Increased lysis of RBC Increased plasma Hb levels DECREASE IN ATP LEVEL

8. The first anticoagulant preservative was introduced by Rous and Turner in 1916,consisted of a citrate-glucose solution. Rous Turner's solution was used for storage of human blood during the First World War. During the Second World War acidified citrate dextrose (ACD) solution was introduced . HISTORY Blood banking was born of wars

9. In 1957 Gibson developed an improved preservative of citrate-phosphate-dextrose (CPD), which was less acidic and maintained 2,3-BPG level better than in ACD solution. In 1978 citrate-phosphate-dextrose with adenine (CPDA-1) preservative was developed. The addition of adenine improved the synthesis of adenosine triphosphate (ATP) in the stored blood, which prolonged the storage of blood/red cells at 2-4 °C to 35 days

10. ACD(acidified citrate, dextrose) CPD(citrate , phosphate , dextrose) CPDA(citrate, phosphate , dextrose , adenine) BLOOD PRESERVATIVES USED ARE

11. Citrate Calcium chelator , prevents coagulation and retards glycolysis . Citrate toxicity - hypocalcaemia and hypomagnesaemia which can result in myocardial depression or coagulopathy . Considered to be a cause of cardiac arrythmia in massive transfusion. Management -   Slowing or temporarily stopping the transfusion allows citrate to be metabolised. Replacement therapy may be required for symptomatic hypocalcaemia or hypomagnesaemia. Action of ingredients of preservatives

12. Adenine- substrate for ATP synthesis, extends the shelf life of red cells to 42 days as RBC has ATP dependant cytoskeleton. Sodium di phosphate – act as buffer and thus prevents fall in pH . Dextrose - improves red cell viability by providing energy for ATP synthesis decreases rate of hydrolysis of phosphorus. Citric acid- prevents glucose caramalization during autoclaving provides optimal pH with citrate for red cells.

13. Acid citrate dextrose (ACD): Composition – Tri-sodium citrate- 22 gm Citric acid (monohydrate) -8.0gm Dextrose (monohydrate)-24.6 gm Distilled water upto1litre For each 100 ml of blood, 15 ml of the ACD solution is needed

14. Advantages of ACD- Preserves ATP level prevents haemolysis maintains pH Shelf life of whole blood / red cells in ACD = 21 days > 70% transfused cells viable after 24 hours However level of 2,3 BPG lost early within first week

15. Citrate phosphate dextrose (CPD) CPD is a modified ACD solution that is slightly less acidic and therefore improves the preservation of 2,3-BPG. Composition - Tri-sodium citrate- 26.30 gm Citric acid- 3.27gm Dextrose- 25.50gm Sodium phosphate-2.22gm Distilled water upto1litre For each 100 ml of blood 14 ml of CPD is added.

16. Avantages of CPD Solution – Better maintenance of 2,3 BPG decreases acidosis improves ATP synthesis Shelf-life of whole blood in CPD = 21 days

17. Citrate Phosphate Dextrose Adenine (CPDA): Adenine - helps maintain high ATP levels ATP is associated with red cell viability .Loss of ATP causes increase in cellular rigidity & decrease in red cell membrane integrity & deformability. A decrease in ATP allows the leak of Na & K through red cell membrane . S helf -life = 35 days

18. Properties of whole blood and packed RBC after 35 days of storage in CPDA Variable 0 day Whole blood Packed cell pH 7.55 6.73 6.71 Plasma haemoglobin (mg/dl) 0.50 46 246 Plasma potassium( mEq /l) 4.20 17.20 76 Plasma sodium( mEq /l) 169.00 153 122 Blood dextrose(mg/dl) 440.00 282 84 2,3 DPG 13.20 1 1 Percent survival - 79 71

19. Inhibits coagulation by inactivating factor Xa , IXa,XIa,plasmin potentiates action of antithrombinIII Dose for anticoagulation-0.5 to 2.0IU/ml It has no preservative property and its anticoagulant effects are neutralized by plasma To be transfused in 24 hrs HEPARIN

20. Sodium salts act as chelating agents and prevent coagulation by binding with Ca Toxic and damages platelets Not used in blood transfusion Used for preserving blood samples only ETHYLENE DIAMINO TETRA AETIC ACID(EDTA)

21. Traditional preservatives were put into use when whole blood was the major product. With the advent of component therapy, use of red cells increased , and so the need of different preservative methods . These include- 1. Optimal additive solutions 2. Rejuvenation solutions 3. Red cell freezing Current trends in blood preservation research

22. Normally 40% of adenine and glucose present in the whole blood, was removed in preparation of packed RBC and thus there was decrease in its viability particularly in last 2 weeks of storage. RBC became more viscous and difficult to infuse in emergency situation However preparation of concentrates of haematocrit less than 80% by allowing adequate plasma to remain, lead to lower plasma yields affecting FFP and Cryoprecipitate production. Optimal Additive Solution (OAS)

23. Thus use of additive solutions for preparation of packed RBC allowed recovery of maximum amount of plasma for preparation of FFP and cryoprecipitate.

24. Different types of additive systems are now in use - (1) Adsol - ( AS-1 ) (2) Nutricel - (AS-3) (3) Optisol - (AS-5) OAS are added to the red cells after separating them from plasma.

25. AS-1 AS-3 AS-5 Sodium citrate - 588 mg - Monobasic sodium phosphate - 276 mg - Citric acid - 42 mg - Dextrose 2.20 g l.l0 g 900 mg Adenine 27 mg 30 mg 30 mg Mannitol 750 mg 525 mg Sodium chloride 900 mg 410 mg 877 mg Vol 100 ml 100 ml 100 ml Primary bag anticoagulant CPD CPD2 CPD COMPOSITION OF ADDITIVE SOLUTIONS

26. The advantages of using optimal additive solution to red cells are - Provide the red cells with adequate nutrients. Better storage condition for red cell preparation and lowering of viscosity for ease of transfusion. Increased yield of plasma for plasma fractionation. Removal of unwanted buffy coat Avoid unnecessary transfusion of plasma Mannitol acts as membrane stabilizer and reduces hemolysis to acceptable levels

27. Additive system increase the level of ATP and red cells, extending the shelf-life of the red cells to 42 days. More than 80% red cells survive in circulation 24 hours after transfusion of blood stored for 42 days.

28. Disadvantage of additive solutions Additive solutions do not maintain 2,3BPG throughout the storage time .Therefore , blood stored in additive solutions is not given routinely to newborn infants.

29. PURPOSE : To increase levels of 2,3 BPG and ATP in stored red cells. Added at any time between 3 days post collection and three days after expiration of red cells . Consists of – phosphate inosine glucose pyruvate adenine Rejuvenation process is expensive and consumes time and is rarely used. (in autologous donations/rare blood groups) REJUVENATION SOLUTIONS

30. Frozen red cells can be stored for 10 yrs but freezing damages red cells due to the intracellular ice formation & hypertonicity . To counter this glycerol is added. Glycerol prevent freezing injury in human red cells & red cells mixed with glycerol could be frozen without damage. RED CELLS – FROZEN STATE

31. Two concentrations of glycerol are used- High glycerol(40% w/v) Low glycerol(20% w/v) Most blood banks use high glycerol technique Generally cells are glycerolized and frozen within 6days of collection of blood in CPDA but RBCs in additive solution can be frozen upto 42days.

32. RBCs frozen at high glycerol can be stored for 10 year. Those frozen at low glycerol can be stored for 3 year. Frozen RBC need to be deglycerolized before transfusion. Deglycerolized for 24 hr.

33. High cost Short post thawing shelf life(24 hrs) Increased red cell loss in processing Increased processing time in emergency DISADVANTAGES

34. Preservation of viable & functional platelets depends on – Temperature – should be stored at 22 – 24 C with continuous gentle agitation in platelet incubator & agitator. pH – should be above 6. Plastic bag – maintenance of pH & function of platelets depends on permeability of storage bag to oxygen & CO2. 50 to 60 ml of plasma is needed for storage as it provides bicarbonate buffers inhibitors of coagulation activation glucose as metabolic substrate PLATELET PRESERVATION

35. Platelets should be prepared within eight hours of phlebotomy and stored at 20-24 C with continuous agitation, to prevent aggregation which can result in loss of viability. Platelets are stored in large flat bags with a high surface-to-volume ratio ,on agitators to facilitate oxygen diffusion. If there is no agitation for more than 24 hours, the bag contents become hypoxic and the metabolism shifts to anaerobic glycolysis so that the contents become acidotic and the platelets lose their function.

36. Synthetic mediums to replace significant portion of plasma volume PRIMARY INGREDIENTS 1.Citrate- prevents activation of anticoagulation 2.Acetate- serves as substrate for activation of anticoagulation 3.Sodium chloride- isotonicity and osmotic strength 4.Phosphate- stimulation of glycolysis and maintenance of physiologic pH 5.magnesium/potassium- decreased platelet activation , improves morphology score , decreased lactate production PLATELET ADDITIVE SOLUTIONS

37. Reduction of allergic reactions and febrile transfusion reactions Facilitate ABO incompatible platelet transfusion Plasma not used can be used for other uses Support 7days of platelet storage Improved efficacy of platelet collection ADVANTAGES

38. Shelf life of FFP is 12 months at – 18 C or lower. After thawing, FFP can be stored at 2 – 6 C for 12 hrs before transfusion. If FFP can not be used within 1 yr or thawed plasma is not used within 12 hrs it is re designated as single donor plasma which can be stored further for 4 yrs at – 18 C or lower. FRESH FROZEN PLASMA

39. Cryoprecipitate is prepared from plasma. It contains fibrinogen ,von Willebrand factor and factor VIII. Cryoprecipitate can be stored for 12 months at – 18 C or lower. Thawed cryoprecipate can be stored for 6 hrs at 2 – 6 C & pooled cryoprecipitate kept at 2 – 6 C should be used within 4 hrs. CRYOPRECIPITATE

40. One of the major factors important in viability of transfused red cells are plastic bags used for storage They should be sufficiently susceptible to co2 so as to maintain higher pH during storage Currently blood is stored in bags made of PVC and DHEP PLASTIC BAGS

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