Storage and preservation of the blood.pptx

in the blood bank

1. Preparation, preservation, and Storage of blood and its components Dr. Amany M. Elshamy Lecturer of Biochemistry and Molecular Diagnostics PH.D of Biochemistry and Molecular biology MLS, AUC, Cairo

2. Outline

3. Introduction The first authentic blood preservative was developed by two English scientists, Loutit and Mollison in the year 1943 and it was known as “Acid Citrate Dextrose (ACD)”.

4. Introduction Sterile collection systems must be used for the collection of blood and blood components. These must be used by the instructions of the manufacturer. A check must be made before use to ensure that the collection system employed has not been damaged or contaminated and that it is appropriate for the intended collection procedure. Defects in blood bags must be reported to the supplier and be subjected to trend analysis. Component labeling and information

5. Component labeling and information Before use, all blood components must be labelled with relevant identity information. The type of label to be used, as well as the labelling methodology, must be established in written procedures. Critical information must be provided in machine-readable format to eliminate transcription errors..

6. Labelling The labelling must comply with the relevant national legislation and international agreements. The following information must be shown on the label or contained in the component information leaflet, as appropriate: • the producer’s identification; • the unique identity number; • the name of the blood component; • the ABO and RhD groups; • blood group phenotypes other than ABO and RhD (optional); • the date of donation; • the date of expiry; • the name of the anti-coagulant solution;

7. Biochemical changes in the stored blood RBC The viability of the red cells depends on the 2-3-DPG and ATP levels. The 2-3-DPG level falls more rapidly than the ATP in the stored blood . The oxygen-releasing capacity of the hemoglobin is directly proportional to the 2-3-DPG leve l. If the 2-3-DPG level falls, less oxygen is released to the tissues. The fall in pH of stored blood also affects 2-3-DPG level. The other changes which take place in RBC are: • RBC changes its shape from discoid to spherical • Osmotic fragility is increased • There is a loss of red cell membrane lipids . Note: 2,3-DPG is the most abundant phosphate compound in the red cell.

8. Biochemical changes in the stored blood W BC The WBC becomes nonfunctional after 24 hours of storage , but they retain their antigenic properties and are quite capable of causing non-hemolytic febrile transfusion reactions in the recipients. Platelets The platelets stored at 4-6°C lose their hemostatic properties within 48 hours. Coagulation factors The coagulation factors, mainly, V and VIII lose their activity within 72 hours of storage.

9. Biochemical changes in the stored blood Electrolytes The most important biochemical change in the stored blood is the loss of potassium from the red cells and the intake of sodium from plasma . The choice of the preservative depends on the percentage of “posttransfusion survival of red cells” and its storage period . The higher the post-transfusion survival and longer the storage period, the better the preservative.

10. Preservative solutions

11. Preservation solutions Preservation solutions contain impermeant agents, osmotic molecules that prevent cell swelling,  purine nucleotide  precursors (adenine, phosphates, ribose) to sustain a low metabolic rate, antioxidants (glutathione, vitamin E),  enzyme inhibitors , buffers (e.g., HCO 3 ) and electrolytes mimicking intracellular (e.g., UW solution) or extracellular (e.g., IGL-1 solution) fluids.

12. Acid citrate dextrose (ACD). Citrate phosphate dextrose (CPD). Citrate phosphate dextrose-adenine (CPDA-1) Preservation solutions

13. Acid citrate dextrose (ACD) The ACD is hardly used as a blood preservative anymore in any modern setup, because of its shorter storage time (21 days) and poor viability of cells (70%). It was used with the glass bottles.

14. Citrate phosphate dextrose (CPD) It was discovered by Gibson, et al that the addition of phosphate in the ACD anticoagulant increases the post-transfusion survival of cells to 80% compared to ACDs 70% after 21 days of storage. The composition of CPD solution is given below: • Trisodium citrate (dihydrate) 26.30 gm • Sodium dihydrogen phosphate 2.28 gm • Dextrose 25.50 gm • Citric acid 3.27 gm • Distilled water 1.0 L The pH of the solution is 5.6-5.8.

15. Citrate phosphate dextrose (CPD) The citrate in the solution acts as the anticoagulant (0.2%) citrate can prevent clotting of 2.5 liters of blood. Phosphate helps in maintaining the pH of the blood. D extrose provides nourishment to the cells and helps in the synthesis of ATP . The post-transfusion survival of cells depends on ATPs.

16. Citrate phosphate dextrose-adenine (CPDA-1) It was observed by Simon in 1962 that the addition of adenine to the CPD solution increases the post-transfusion survival of cells to 80% and the storage period to 35 days . The ATP concentration is higher and the viability is higher in adenine blood. The composition of CPDA -1 is given below: • Trisodium citrate (dihydrate) 26.30 gm • Sodium dihydrogen phosphate 2.22 gm Dextrose (monohydrate) 31.8 gm • Adenine 0.275 gm • Citric acid 3.27 gm • Distilled water 1 L

17. Citrate phosphate dextrose-adenine (CPDA-1) The ratio of preservative to blood is 14 ml of CPDA-1 solution for 100 ml of blood. The amount of preservative present in 350 ml and 450 ml capacity bags is 49 ml and 63 ml respectively. The other preservative CPDA-2 in which the amount of dextrose is increased to 44.6 gm, improves the viability of cells further.

18. A dditive solution

19. Benefits of RBC Additive Solutions Extends the shelf-life of RBCs to 42 days b y adding nutrients • Allows for the harvesting of more plasma and platelets from the unit • Produces an RBC concentrate of lower viscosity that is easier to infuse

20. A dditive solution An additive solution may be defined as a solution packaged in a blood bag set which: (I) is contained separately in a satellite bag, but is attached by tubing to the primary CPD-containing bag; (2) is an isotonic solution containing some or all of the nutrients needed for extended red cell storage.

21. A dditive solution The commonly used additive solutions are: CPD-SAG. CPD-SAGM. CPD- Adsol .

22. A dditive solution CPD-SAGM The SAGM stands for Saline-Adenine-Glucose-Mannitol. The four-bag system introduced by Hogman , et al in 1978, contains a preservative solution in two bags. Bag 1 contains 63 ml of CPD solution while bag 2 contains 100 ml of SAGM solution.

23. A dditive solution CPD-SAGM 450 ml of blood is collected in the primary bag containing CPD solution, the plasma is expressed after centrifugation to the empty bag and then the 100 ml of the SAGM solution is expressed in the primary bag containing red cells. The red cells can be stored for 42 days in SAGM. The post-transfusion survival of cells in SAGM is more than 80%.

24. A dditive solution CPD- Adsol The composition of CPD- Adsol is the same as of CPD-SAGM, except that, it contains higher concentrations of glucose, adenine, and mannitol, which improves the viability of cells considerably.

25. The preparation, use and quality assurance of blood components

26. 1. Preparation of RBCs For the preparation of Red Cell components , plasma is removed from Whole Blood by centrifugation. Requirements and quality control .

27. 2. Preparation of Red Cells, Leucocyte-Depleted( Red Cells, LD . ) Red Cells, Leucocyte-Depleted (LD) is a red cell component derived from Whole Blood donation, Red Cells or Red Cells, BCR by removing the leucocytes. Red Cells, LD contains a minimum haemoglobin content of 40 g. The hematocrit is 0.65-0.75. Red Cells, LD contains less than 1.0 × 10 6 leucocytes. Preparation Generally a filtration technique is used to produce Red Cells, LD . Leucocyte depletion within 48 hours after donation is the standard. Red Cells, LD can be produced: • by leucocyte filtration of Whole Blood , with subsequent centrifugation and removal of the plasma; • by leucocyte filtration of a red cell component.

28. 3. Red Cells, Apheresis Definition and properties Red Cells, Apheresis ( Aph ) is a red cell component obtained by apheresis of a single donor using automated cell-separation equipment. For preparation of Red Cells, Aph , Whole Blood is removed by an appropriate apheresis machine from the donor and anti-coagulated with a citrate-containing solution. The plasma is returned to the donor. Either one or two units of Red Cells, Aph can be collected during a single procedure.

29. 4. Platelets, Recovered, Single Unit (Rec, SU) Platelets, Recovered, Single Unit (Rec, SU) is a platelet component derived from a single Whole Blood donation. It contains the majority of the original Whole Blood platelet content, suspended in plasma. Platelets, Rec, SU contains more than 60 × 10 9 platelets. Platelets, Rec, SU contains up to 0.2 × 10 9 leucocytes if prepared by the platelet-rich plasma method, and up to 0.05 × 10 9 leucocytes if prepared by the buffy coat method. Platelets, Rec, SU can be used for neonatal and infant transfusion . In order to achieve a ‘standard adult dose’, 4 to 6 units of Platelets , Rec, SU have to be transfused.

30. Preparation from platelet-rich plasma (PRP) A unit of Whole Blood , stored for up to 24 hours in conditions validated to maintain the temperature between + 20 °C and + 24 °C, is centrifuged so that an optimal number of platelets remain in the plasma and the number of leucocytes and red cells are reduced to a defined level. Platelets from PRP are sedimented by hard-spin centrifugation; the supernatant platelet-poor plasma is removed, leaving 50-70 mL of it with the platelets. The platelets are allowed to disaggregate and are then re-suspended in the remnant plasma.

31. Preparation from buffy coat A Whole Blood unit, stored for up to 24 hours in conditions validated to maintain the temperature between + 20 and + 24 °C, is centrifuged so that platelets are primarily sedimented to the buffy coat layer together with the leucocytes. The buffy coat is separated and processed further to obtain a platelet concentrate. Single buffy coats diluted with plasma are centrifuged so that the platelets remain in the supernatant, but red cells and leucocytes are sedimented to the bottom of the bag.

32. 5. Platelets, Apheresis Definition and properties Platelets, Apheresis ( Aph ) is a component obtained by platelet apheresis of a single donor using automated cell separation equipment, which contains platelets in a therapeutically effective dose suspended in plasma. Platelets, Aph contains a minimum content of 2 × 10 11 platelets. Platelets, Aph contains a maximum leucocyte content of 0.3 × 10 9 cells.

33. 5. Platelets, Apheresis Preparation For preparation of Platelets, Aph , Whole Blood is removed from the donor by the apheresis machine, anti-coagulated with a citrate solution and then the platelets are harvested. For use in neonates and infants, Platelets, Aph can be divided into satellite units under sterile conditions.

34. Platelets, Apheresis

35. 6. Plasma, Fresh Frozen ( FFP) Definition and properties Plasma, Fresh Frozen (FFP) is a component for transfusion or for fractionation, prepared either from Whole Blood or from plasma collected by apheresis, frozen within a period of time and to temperature that adequately maintains the labile coagulation factors in a functional state. It must contain, on average, 70% or more of the value of the freshly collected plasma unit and at least similar quantities of the other labile coagulation factors and naturally occurring inhibitors.

36. 6. Preparation of Plasma, Fresh Frozen ( FFP) From Whole Blood Plasma is separated from Whole Blood that has been collected using a blood bag with integral transfer packs employing hard-spin centrifugation and collected preferably within 6 hours. Alternatively, plasma may be separated from platelet-rich plasma. Plasma may also be separated from Whole Blood that, immediately after donation, has been cooled rapidly by a special device validated to maintain the temperature between + 20 °C and + 24 °C and is held at that temperature for up to 24 hours. It is prepared from a single-pack Whole Blood donation, adequate sterility precautions must be adopted.

37. 6. Plasma, Fresh Frozen ( FFP) By apheresis FFP may be collected by apheresis. The freezing process must commence preferably within 6 hours of collection and not more than 18 hours after completion of the procedure if the unit is refrigerated. Freezing must take place in a system that allows complete freezing within one hour to a temperature below – 30 °C.

38. 7. Cryoprecipitate Definition and properties Cryoprecipitate is a component containing the cryoglobulin fraction of plasma obtained by further processing of Plasma, Fresh Frozen and then concentrated. It contains a major portion of the Factor VIII, von Willebrand factor, fibrinogen, Factor XIII and fibronectin present in freshly drawn and separated plasma.

39. 7. Preparation of Cryoprecipitate Plasma, Fresh Frozen is thawed, either overnight between + 2 to + 6 °C or by the rapid thaw-siphon thaw technique. After thawing, the component is re-centrifuged using a hard spin at the same temperature. The supernatant cryoprecipitate-poor plasma is then partially removed. The sedimented cryoprecipitate is then rapidly frozen.

40. 7. Preparation of Cryoprecipitate Preparation When Cryoprecipitate is prepared from Whole Blood -derived plasma, the maximal final volume of the component is 40 mL. Alternatively, Plasma, Fresh Frozen obtained by apheresis may be used as the starting material and the final component can be prepared using the same freezing/thawing/re-freezing technique. Leucocyte depletion of the starting material and/or virus inactivation, and/or quarantine is a requirement in some countries.

41. 7. Cryoprecipitate

42. 8. Granulocytes, Apheresis Definition and properties Granulocytes, Apheresis is a component that contains granulocytes suspended in plasma and is obtained by apheresis of a single donor using automated cell separation equipment.

43. 8. Granulocytes, Apheresis An adult therapeutic dose of Granulocytes, Apheresis contains between 1.5 × 10 8 and 3.0 × 10 8 granulocytes/kg body weight of the designated recipient. Granulocytes, Apheresis has a significant content of red blood cells, lymphocytes, and platelets. Donors of Granulocytes, Apheresis require pre-treatment with corticosteroids and/or growth factors. HLA type is required. Granulocytes, Apheresis must be irradiated.

44. Storage

45. RBCs storage Red Cells must be kept at a controlled temperature between +2 °C and + 6 °C. The storage time depends on the anti coagulant/preservative solution used. For example, the storage time is 35 days in CPDA-1. Validated transport systems must ensure that at no time during a maximum transit time of 24 hours did the temperature exceed + 10 °C.

46. Long-term storage of red cells The red cells in a frozen state can be stored for years. Freezing damages red cells by intracellular ice formations and hypertonicity. This can be prevented by the addition of glycerol. The glycerol limits the ice formation and provides a liquid phase for cooling of salts, by permeating the red cells easily during freezing.

47. Long-term storage of red cells ( Glycerolisation ) High glycerol solution 40% w/v concentration of glycerol is known as high glycerol solution. The red cells are frozen at –180°C over a period of 30 minutes by mechanical refrigeration and then stored at –40 to –50°C for 3 years. Low glycerol solution The 20% w/v concentration of glycerol is known as low glycerol solution. The red cells are frozen at –190°C using liquid nitrogen for 2-3 minutes and then stored in the gas phase of liquid nitrogen for 3 years .

48. Thawing and deglycerolisation Since the red cells are stored in frozen state, so, they must be brought to body temperature. The process is carried out by keeping the frozen red cells in a water bath at 37°C for 10 minutes. The procedure is known as Thawing. The glycerol must be removed properly ( Glycerol can cause hemolysis) . The principle of deglycerolisation is that frozen red cells are kept first in hypertonic solution, and then moved to less hypertonic and finally to isotonic solution. Red cells from a donor with sickle cell trait form an insoluble jelly-like mass during deglycerolization .

49. Procedure for deglycerolisation • The 40% w/v glycerolised red cells are diluted in 12% sodium chloride buffered to pH 7.2 with 0.15% disodium phosphate and equilibrated for 5 minutes. Wash the red cells in 2 litres of 1.6 % of sodium chloride solution buffered to pH 7.2 with 0.03 gm% of disodium phosphate. Finally wash the red cells in isotonic glucose solution, which is prepared in 1 litre of 0.9 gm% of sodium chloride solution, containing 0.2 gm% of glucose buffered with 0.0 65 gm% disodium phosphate to a pH of 6.8. The shelf life of deglycerolised blood is 24 hours.

50. Storage of plasma Storage of plasma The fresh frozen plasma (FFP) is stored at – 30°C for 1 year. 36 months at below – 25 °C 3 months at – 18 °C to – 25 °C

51. Storage of platelets The platelets are stored ( in an agitator) at room temperature (RT) 20-25°C for 5 days only. More than 5 days of storage of platelets at RT increases the chance of bacterial contamination of platelets and the loss of platelet quality during storage (known as the platelet storage lesion).

52. Storage of platelets During storage, platelets can become activated, leading to the release of various substances such as adenosine diphosphate (ADP), thromboxane A2, and serotonin . This activation can result in platelet aggregation and the formation of a platelet plug, which is essential for clot formation and hemostasis. Platelet activation is a normal physiological response to injury or inflammation, but it can also occur during storage due to various factors such as temperature changes, exposure to certain chemicals, or prolonged storage time.

53. Storage of Cryoprecipitate The stability of Cryoprecipitate on storage is dependent on the storage temperature. The optimal storage temperature is below – 25 °C. Recommended storage times are: • 36 months at or below – 25 °C; • 3 months at – 18 °C to – 25 °C.

54. Storage and transport of Granulocytes.

55. Qs

56. The correct temperature for shipping RBCs is A. 1 to 6C B. 1 to 10C C. 20 to 24C D. 37C   RBC storage times vary with the anticoagulant/preservative used.  Which of the following is properly paired? A. Citrate-phosphate-dextrose (CPD): 35 days B. Additive solution (AS): 47 days C. Citrate-phosphate-dextrose-adenine (CPDA)-1: 35 days D. Acid-citrate-dextrose (ACD): 35 days

57. w hich of the following represents a change seen in a unit of RBCs stored with CPDA-1 at the end of its shelf life? A. Percentage of viable cells at 24 hours after transfusion decreases to 71% B. Supernatant K+ (potassium ion) concentration decreases C. Supernatant pH increases D. Red cell 2, 3-DPG increases E. Supernatant hemoglobin decreases

58. A unit of RBCs is issued to the floor and returned without being transfused.  How long can blood be out of the refrigerator and still be used for transfusion? A. 10 minutes B. 30 minutes C. 1 hour D. 4 hours

59. The maximal shelf life of irradiated RBCs is : A. 4 hours B. 6 hours C. 24 hours D. 21 days E. 28 days

60. The preferred method for generating leukocyte- reduced RBC components is: A. Thawing and deglycerolizing a frozen unit B. Filtering using a leukocyte-reduction filter C. Irradiation D. Centrifugation E. Washing

61. The most common high concentration of glycerol used in the U.S. for freezing RBCs is: A. 5% B. 10% C. 20% D. 40%

62. The rationale for deglycerolizing frozen RBCs with extensive washing is: A. Glycerol is not approved by the FDA B. Glycerol is toxic to kidneys C. Glycerol can cause hemolysis D. Glycerol can cause anaphylaxis

63. Which of the following choices explains why a unit of blood may form an insoluble jelly-like mass during deglycerolization ? A. Inadequate deglycerolization B. Bacterial contamination C. Insufficient anticoagulant D. Inadvertent use of hypotonic saline for washing E. Red cells from a donor with sickle cell trait

64. According to the AABB standards, the maximum allowable shelf-life of platelets without gentle agitation is: A. 1 hour B. 4 hours C. 8 hours D. 24 hours

65. According to AABB standards, 90% of the units of random-donor platelets prepared from whole blood should contain a minimum of _______ platelets per unit. A. 5.5 x 10^ 9 B. 5.5 x 10 ^ 10 C. 5.5 x 10 ^11 D. 3 x 10 ^ 10 E. 3 x 10 ^ 11

66. The minimum acceptable pH of platelet units at the end of the storage period is: A. 4.2 B. 5.2 C. 6.2 D. 7.2 Which of the following is a change associated with platelet storage? A. Decreased H+ concentration B. Platelet activation C. Change in shape from round to discoid D. Increased swirling effect

67. Transfusion of one platelet concentrate  ( ie the platelets present in one whole blood donation) into a hematologically stable adult of average size with no history of transfusion and/or pregnancy is expected to increase the platelet count by: A. 1000 to 5000 / uL B. 3000 to 5000 / uL C. 3000 to 12000 / uL D. 5000 to 10000/ uL

68. FFP that has been thawed and is being stored at 1 to 6 C should be transfused within A. 4 hours B. 6 hours C. 12 hours D. 18 hours E. 24 hours Which of the following is true? A. To prepare FFP plasma must be separated from red cells within 24 hours B. If an additive solution is used, the expiration date for RBCs stored at 1 to 6 C is 42 days after phlebotomy C. To prepare Cryo FFP is thawed at 20 to 24 C D. Platelets derived from a unit of whole blood must contain 3 x 10 ^ 11 platelets in 75% of units released

69. A group B Rh-positive patient requires FFP.  Group B FFP is not available.  The substitute component of choice is: A. Group O FFP B. Group A, Rh negative cryo C. Group A FFP D. Group AB FFP After thawing cryo should be stored at: A. 42C B. 37C C. 20 to 24 C D. 1 to 6 C

70. According to AABB standards each bag of cryo must contain a minimum of how many Internation Units of Factor VIII? A. 70 B. 80 C. 100 D. 120 Cryo contains which of the following? A. Factor XI B. Protein C C. Protein S D. Factor XIII

71. Which is true regarding cryo ? A. Is prepared from FFP thawed at 20 to 24C B. Once thawed is stored at 1 to 6 C and given within 6 hours of thawing or 4 hours of pooling C. It is prepared by filtering thawed FFP at 1 to 6 C D. It is prepared by centrifuging thawed FFP at 1 to 6 C

72. Granulocytes are stored at A. 1 to 6 C without continuous agitation B. 1 to 6 C with continuous agitation C. 20 to 24 C without continuous agitation D. 20 to 24 C out continuous agitation The shelf life of granulocytes is: A. 4 hours B. 6 hours C. 12 hours D. 24 hours