1. Components Preparation By: Wajnat Tounsi

2. Why preserve blood?! To provide patients who need blood components transfusion with VIABILE products. RBCs viability is a measure of in-vivo RBC survival following transfusion. 75% of transfused cells must remain viable for 24hours post transfusion.

3. Red Cells Biology Normal red cells function and survival depends on: 1.Normal chemical composition and structure of the RBC membrane 2.Hb structure and function 3.RBCs metabolism

4. Cell Membrane RBC has a semipermeable membrane Consist of two bilayer sheets: integral and peripheral The external is rich in glycolipids and choline phospholipids Cell membrane plays a crucial role in permeability and deformability

5. For RBC to function normally it needs to maintain it’s permeability, deformability, and flexibility. Decrease in adenosine triphosphate (ATP) level lead to loss of membrane deformability Increase in Ca +2 deposition on membrane lead to loss of flexibility Cell Membrane

6. RBC membrane is permeable to water and anions (Cl -, HCO 3 - ) RBC membrane is relatively impermeable to cations (Na +, K + ) When ATP is lost, Calcium and sodium accumulate in the cell, potassium and water are lost leading to a rigid cell Cell Membrane

7. Red cell has no nucleus. The main ATP source is through the break down of glucose (glycolysis) Cell metabolic

8. Hb Structure and Function Hb main function is to transport gases 2,3-diphosphoglycerate (2,3-DPG) is an important control for Hb affinity to oxygen. When partial pressure of oxygen (PO 2 ) is 100 mm Hg, Hb is 100% saturated with oxygen When PO 2 is 40 mm Hg, Hb-oxygen saturation drops to 75%, releasing 25% oxygen to the cells

9. Normal association curve depends on (H+, CO2, phosphate and 2,3-DPG). “Shift to the left” occur when Hb has high affinity to oxygen, release only 12% of oxygen at 40 mm Hg. “Shift to the right” occur when Hb has low oxygen, release 50% of oxygen at 40 mm Hg. Hb Structure and Function

10. Hb-Oxygen Association Curve

11. Anticoagulant Solution used to maintain the viability of red cells during storage to the time of transfusion. It is important to maintain ATP and 2,3-DPG levels Preservatives

12. Acid-Citrate-Dextrose (ACD) – Storage time is 21 days Citrate-phosphate-dextrose (CPD) – Storage time is 21 days Citrate-phosphate-double dextrose (CP2D) – Storage time is 21 days Citrate-phosphate-dextrose-adenine-1 (CPDA-1) – The current used one – Storage time is 35 days

13. Action of ingredients of anticoagulant solution : – Citrate: bind to calcium ion to prevent blood clot – Phosphate: maintain the pH – Glucose : ATP generation – Dextrose: maintain ATP level – Adenine: support ATP level, extend life of RBCs Preservatives

14. Additive Solutions 100 ml solution added to the PRBCs after removing the plasma with/without platelets. Removing plasma during components preparation remove nutrients that RBCs need. PRBCs have high hematocrit (80%) and viscosity which can be heard to transfuse. Additive solution reduce the hematocrit to (50- 60%) All additive extend the shelf life of RBCs to 42 days.

15. AS-1: contain SAGM with CPD Nutricel (AS-3): contain SAG with CP2D Optisol (AS-5): contain SAGM with CPD Additive Solutions

16. SAGM Saline: provides isotonicity Adenine: maintain ATP level Glucose: support cells metabolism Mannitol: reduce cell lysis The stander additive solution used

17. Biochemical Changes Decrease in PH Lactic acid accumulation Decrease in glucose consumption Decrease in ATP level Loss of RBCs function (shift to the left) Anticoagulant and preservatives minimize biochemical changes and increase shelf life

18. Blood Components

19. RBC Platelets Fresh frozen plasma cryoprecipitate

20. Blood Components Light spin: short time, low RPM Heavy spin: longer spin, high RPM

21. PRBCs Whole blood must be processed through the first 8 hours after collection Prepared by separating the plasma with final hematocrit = 80% or less 1 unit increases Hb 1 g/dL, and Hct 3% All PRBC unit is stored (1°-6° C) for either (21, 35, 42) days depending on the anticoagulant and the additive solution used.

22. Weight and balance each unit. Load unit into the swinging bucket apparatus. Centrifuge using heavy spin (5000 rpm) for 5 min. After that, place the unit carefully onto an expressor and release the spring Remove (230-256 g) plasma to one satellite bag to create about 80% hematocrit Add SAGM if present PRBCs

23. Weight and balance units and centrifuge

24. Separated whole blood Express plasma to satellite bag

25. Seal tubes and separate Packed Red Blood Cells Store units at 1°-6° C

26. Separate the bag by heat sealer PRBC unit is stored at (1°-6° C). PRBCs

27. Indication Anemia Blood loss Trauma Oncology patients Cardiac surgery Renal disease

28. RBC Aliquots RBC transfused to neonates at the age of (0-4) months. (10-25) ml of PRBC is transfused

29. Irradiated RBCs Gamma irradiation used to deplete T cells. T cells proliferation responsible for GVHD Stored for 28 days after the irradiation Indication include: – Immunocompromised patient – Intrauterine transfusion – Bone marrow, or stem cells receiving patient – HLA-matched donor unit – Patient who had fibril transfusion reaction

30. Leukoreduced RBCs Leukocytes are linked to fibril transfusion reaction, transfusion related lung injury, transmission of (Epstein-Barr virus, HTLV, CMV) WBC count in the unit is less than 5 x 10 6 Blood pass through filter, retention of 85 % of RBCs Either: prestorage, or poststorage reduction.

31. Prestorage: filter used to remove 99.9% of leukocytes. Filter is made out of layers of synthetic nonwoven fibers. The filter allow RBCs to pass through and trap WBCs and platelets. Leukoreduced RBCs

32. There are three methods for prestorage reduction: 1. In line filter attached to the WB unit, which then filtered by gravity. Platelets and plasma prepared afterward. 2. RBCs are Filtered after the preparation of plasma. For the first two, platelets can NOT be prepared. 3. A sterile docking device is used to attach filter to a unit of RBCs. Leukoreduced RBCs

33. Poststorage reduction: Leukocytes are removed before issuing blood Or at bedside before transfusion Leukoreduced RBCs

34. Frozen, Deglycerolized RBCs Frozen RBCs can be stored for up to 10 years. For rare phenotype, or autologous donation Deglycerolized product is platelets, plasma, and leukocytes free Cryoprotective agent can be either penetrating and non-penetrating.

35. penetrating agent is a small molecule cross the membrane into cytoplasm and prevent water from leaving the cell when ice is forming which prevent dehydration. For e.g. glycerol Non-penetrating agent is a larger molecule that form a shell around the cell to prevent water loss. for e.g. hydroxyethyl starch. Frozen, Deglycerolized RBCs

36. The unit thawing requires 30 min in a water path at 37° C. Deglycerolize: wash to remove cryoprotective agent Frozen, Deglycerolized RBCs

37. Platelet Concentrates Can be prepared either by apheresis (single donor) or during the making of PRBC

38. Indication include: Bleeding thrombocytopenic patient (<50,000/μl) Cancer patient (chemotherapy & radiation) Not recommended for DIC Platelet count in a unit should be at least 5.5x10 10 Platelet concentrate is stored at (20°-24° C) with continuous agitation for 5 days A unit contain 50-70 ml of plasma to maintain pH≥6.2 Platelet Concentrates

39. Preparation Maintain Centrifuge temperature at 20° to 24° C during preparation Centrifuge the WB on light speed (3200 rpm) for 2-3 min. The spin will separate the WB to PRBCs and platelet- riche plasma (PRP) Express the PRP to the satellite bag. Enough plasma should remain within the RBCs to main 80 % hematocrit Seal the tubing between RBC and plasma Re-centrifuge PRP at 22 C on heavy spin (3600 rpm) for 5 min.

40. Preparation The platelets will be separated from plasma Express most of plasma to the second satellite bag leaving 50 to 70 ml to maintain pH Seal the tubes Plasma can be stored as Fresh Frozen Plasma (FFP) Let platelets to rest on the counter for 1 hour Platelet units can be pooled into one bag (6-8 units). Pooled platelets must be transfused with 4 hours

41. After second centrifugation, express plasma to the satellite bag. Leave 50 - 70 ml of plasma to maintain pH. After allowing the platelets to rest for one hour, suspend the platelets back to the plasma. Store at room temp. with continuous agitation for 5 days.

42. Plasma (FFP, PF24) Fresh Frozen Plasma (FFP) contains maximum levels of labile and nonlabile clotting factors about 1IU/ml FFP is frozen within 8 hours after collection of whole blood PF24 contains all stable proteins PF24 prepared within 8-24 hours after collection Plasma is stored at -18° C for 1 year Or -65° C for 7 years

43. Plasma is thawed at 30°-37° C temp. If water bath is used, a plastic lining is used to prevent port contamination. After being thawed completely, store at 1°-6° C until time of transfusion, but not longer than 24 hours. A unit should contains 150-250 ml of plasma, 400 mg of fibrinogen, and 1 unit/ ml of each stable factors, 1 unit/ ml of factor V and VIII. Plasma (FFP, PF24)

44. Indication: – Bleeding patient with multiple factor deficiency – Massive trauma – Surgery – Liver disease – DIC Plasma (FFP, PF24)

45. Preparation FFP/PF24 prepared from whole blood as a part of platelet concentrates preparation After the platelet concentrates has been separated: Weight the plasma and record the volume Place plasma in protective container and freeze The label must contain all the standard information

46. Plasma/ Liquid Plasma Can be prepared from whole blood Or as by-product of platelet concentrates or cryoprecipitate production Used as volume expander Can be prepared during storage or up to 5 days after expiration of whole blood Store for up to 5 years at -18° C At liquid state store at 1°-6° C for 5 days

47. Cryoprecipitated Antihemophilic Factor Cryoprecipitate is the cold-precipitated concentration of factor VIII. Antihemophilic Factor (AHF) Contains at least 80 units of AHF and 150 mg of fibrinogen Stored for up to 12 months in -18° C Must be transfused within 6 hours after thawing and 4 hours after pooling

48. Preparation Platelet concentrates is not made because of the plasma volume required to remain in the PRBCs and platelets will affect the cryoprecipitate production Freeze plasma within 8 hours after collection After completely frozen, start thawing process in the refrigerator at 1°-6° C for 14-16 hours Or thaw in a 4° C water bath Endpoint when plasma becomes slushy

49. Centrifuge plasma at 4° C (heavy spin) Express supernatant plasma to the satellite bag Leave about 10-15 ml of plasma Cryoprecipitate is a small white mass in the bag Separate and freeze cryoprecipitate immediately for 12 months in -18° C preparation time should not exceed one hour. If supernatant plasma frozen, it should be labeled as plasma cryoprecipitate reduced Preparation

50. Component’s Labeling All components must be labeled according to AABB, ISBT 128 All blood bank facilities should have their own unique labeling system Any modification should be identified Serological results ABO and Rh type Expiration date Autologous or volunteer

51. Thank you