Preparations of Blood component, for use.

Describes the methods in detail how to prepare Blood Components.

1. Preparations of Blood components Dr. P K Maharana, Medical Officer Red Cross Blood Center, Bhubaneswar, KIMS

2. Blood & its components.

3. Whole Blood Whole blood contains 450 ± 10 % ml or 350 ± 10%ml of donor blood plus anticoagulant solution. So final volume for 350 ml collection is 350+49= 399 ml ±10% Anticoagulant is used with the name of the product e.g. CPDA-1 Whole Blood. Whole blood has a hematocrit of 30-40 per cent . Stored blood has no functional platelets and no labile coagulation factors V and VIII

4. Components of Blood Blood is a mixture of plasma & cells. The components are : RBC ( PRBC) Plasma ( FFP) Cryoprecipitate Platelet Concentrate (RDP / SDP) .

5. Plasma Blood plasma is a fluid medium in which blood cells are suspended. Plasma carries nutrients to the body’s cells and also flushes waste products to be excreted by the body. It is 55% of total blood volume. The most important components in plasma are water, proteins and clotting factors.

6. How Blood is collected? Normally blood is collected from a suitable donor under proper sterile condition in a blood collecting bag containing required quantity of anticoagulants. Commonly there are three types of bags ; single, double or triple bags. Single Bag for whole blood, double bag for PRBC & plasma, triple bag for all the three components. Depending on the size of bag and amount of anticoagulants present in it, the amount of blood to be collected is decided; it may be either 350ml or 450ml; 350ml bags contains 49ml of anticoagulant @17ml per 100ml. For neonates, infants & children who requires lesser amount can be collected in smaller bags or full bag can be utilize to prepare small bags.

7. Blood Collection Bags Double bags are used for making red cells and plasma only. Triple packs system with two attached bags makes it possible to make red cells, platelet concentrate and fresh frozen plasma. While quad packs system with three attached bags are used for preparing: Red cells, Platelet concentrate, Cryoprecipitate (factor VIII) and Cryo- poor plasma .

8. Different type of collection bags.

9. Anticoagulants in use. Anticoagulants are in use are of two types: CPD & CPDA. C =Citrate P = Phosphate D = Dextrose A = Adenine. Citrate – Binds Calcium prevents clotting . Dextrose – Supports ATP generation by glycolytic pathways. Adenine – Substrate for ATP synthesis.

10. Why Components? Present concept is to transfuse the specific components of the blood instead of transfusing whole blood contrast to earlier practice. A single unit of blood collected can be segregated to different components like red cells , plasma , platelets & cryoprecipitate , which can be of use for four beneficiaries.

11. Additive Solution SGAM solution : Added to RBC, which contains saline , glucose, adenine & manitol . Final Haematocrit in SGAM is around 66%, life is 42 days. ( In non additive CPD the haematocrit is around 80% ).

12. How components are separated? Components are separated from whole on the basis of specific gravity of different components.

13. Different specific gravity of cellular components Due to different specific gravity of cellular components, they can be separated by centrifuging at different centrifugal force (in g) for different time . Component Specific Gravity Whole blood 1.053 Red Cells 1.08 – 1.09 Platelet 1.03 – 1. 04 Plasma 1.02 – 1. 03

14. Centrifuges Blood separation centrifuges tend to make use of either a fixed-angle rotor or a swing-out rotor. The physical force from continuous revolutions pushes the denser, heavier particles to the outer edges of the sample resulting in three layers of different densities : 1. Plasma at the top. 2. A buffy coat : a mixture of ( WBCs , Platelets), between RBC or Plasma. 3. RBCs at the bottom

15. FFP Plasma separated from donor blood is fresh frozen within 24 hours of the donation and the frozen plasma can be kept at -25°C or lower for up to three years. Plasma can also be donated by the  apheresis method .

16. FFP Fresh Frozen Plasma (FFP) is the liquid part of the blood obtained after separation of the cellular part of the blood. The separated liquid part is immediately frozen. FFPs have a volume of 200–300 mL, frozen to a temperature of –25° C within 6 hours, to ensure activity of the coagulation factors. FFPs can be stored for up to one year at a temperature of –25° C. However, prior to administration, FFPs would be needed to be thawed, and need to be used within 30 minutes of thawing. FFPs primarily contain stable clotting factors, albumin and Immunoglobulins. Generally, the dose of FFPs ranges 10–15 mL/kg. The target INR is 1.7, prothrombin time (PT) a activated Prothrombin time (APTT) of less than twice the normal.

17. Q : What is Cryoprecipitate? Ans: It is the cold insoluble portion of plasma remaining after FFP has been thawed.   The volume of cryoprecipitate obtained  is 25-30 ml. Self life is one year . It contains Factor VIII ,Von Willebrand factor, factor XIII and fibrinogen.

18. Cryoprecipitate Cryoprecipitate is the fraction of the liquid part of the blood (plasma), which is undissolved following thawing of the plasma. Cryoprecipitate contains: Fibrinogen (150–300 mg/pack), Factor VIII (80–100 IU/pack), Von Willebrand factor and fibronectin. Similar to FFPs, cryoprecipitate packs can be stored at –25° C for up to one year. The thawed product needs to be used within 6 hours of thawing.

19. How Cryoprecipitate is prepared? Preparation of cryoprecipitate : 1. Plasma is separated from a unit of blood within 6-8 hrs of donation and rapidly frozen within 30 minutes to -30 °C. 2. The frozen plasma is then thawed slowly at below+ 4 degree C  to obtain maximum yield of cryoprecipitate . 3. Cryo is the insoluble portion, or precipitate, that remains when the liquid portion of the plasma drains away. 4 . Finally is stored at -40 degree C or colder . Shelf life of one year..

20. Steps for Preparation of Cryo 1.Whole blood is separated soon after collection to Plasma & Red cells 2 .Gently hang the bag containing the plasma into the freezing solution ensuring that the whole bag is immersed and that the attached satellite bag is placed on the side panels of the bath. Leave in the solution for ten minutes. 3. Remove the frozen plasma, and immediately commence thawing. ( Place frozen bag in a thermostatically controlled water bath at 3° C until solution becomes “slushy” ); approximately 20 - 30 minutes . 4.Immediately spin in a refrigerated centrifuge for 10 minutes at 2000 rpm at 2°- 8° C. Optimum temperature is 3° C. 5. Remove the excess plasma into the empty satellite bag leaving approximately 10 ml with the deposited cryoprecipitate. 6.Detach the excess plasma bag from the cryoprecipitate.

21. Prepare the freezing solution of alcohol (Ethanol) 1. Pour the alcohol into the stainless steel sink. 2. Add dry ice, broken into handsized bits, until the temperature reaches -60 ° C . ( Approximate ratio of 2 kg dry ice to 10 liters of ethanol required to achieve the desired temperature ).

22. Flow Chart for Preparation of Cryoprecipitate

23. PRBC

24. Red cells preparation through Sedimentation: The blood after collection is kept upright in refrigerator at 2-6°C, the red cells settle down and the clear supernatant plasma is transferred into a satellite bag.

25. Centrifugation ( Separation of Plasma & PRBC) 1 . Collect appropriate volume of donor blood in CPDA double or triple bag . 2. Store at 2-6°C till processed. 3. Place bags in the buckets of refrigerated centrifuge and balance the opposite bags accurately . 4. Centrifuge at heavy spin ( 5000 x g ) for 5 minutes at 2-6 degree C. 5 . Express approximately 3/4 of the plasma into the satellite bag . 6. Double seal the tube between primary and satellite bags with plasma. Separate the satellite bag with plasma and keep at -30°C or below. 7. Keep the red cells at 2-6°C.

26. Packed Red Blood Cells in additive solution (Adsol/SAGM): 1 . Collect the appropriate volume of donor blood in primary bag of additive system, consisting of a primary bag containing anticoagulant solution CPD or CP2D attached with at least two satellite bags, one of which is empty and another contains 100 ml of additive solution e.g. Adsol or SAGM. 2 . Store at 2-6oC till processed . 3. Centrifuge at heavy spin as above. 4. Remove most of the supernatant plasma in the empty satellite bag. 5. Add the additive solution to the red cells. 6. Keep the red cells at 2-6°C and plasma at -30°C or below .

27. Preparation of red blood cell concentrates ( Packed Red Blood Cells ) Red blood cells (packed red cells) are prepared by removing most of the plasma from a unit of whole blood. Red cells have higher specific gravity than plasma, the red cells settle in the lower portion of bag due to the gravitational settling ( sedimentation ) or centrifugation. The plasma is transferred into a satellite bag .

28. Expiry Date of PRBC The shelf life of a red blood cell unit is 42 days from collection. The expiry date is documented on the label of each unit. Manipulation of the unit, including washing or irradiation, shortens the shelf life. If the red blood cell unit is opened without the use of a sterile connection device, the shelf life is limited to 24 hours if stored at 1–6°C (or the original expiry date, whichever is sooner), or to 4 hours if stored above 6°C.

29. Rejuvenated Red cells A special solution added to expired RBCs within 3 days of expiry. It restores 2-3 DPG & ATP to pre-storage value . It regains the power to transport oxygen, Expiry 24 hours (if frozen 10yrs).

30. Washed red cells ( W-RBC) RBCs are washed with normal saline to remove the plasma proteins, platelets, white cells, micro aggregates . It can be done through a machine or manually . Normally the RBCs are washed three times. It is mostly recommended for patients with IgA deficiency or with circulating ant-IgA antibodies. It helps to prevent febrile reactions & urticarial rash.

31. Red cells preparations & their contents are: A.) Sedimented red cells: They have a PCV of 60-70 percent, 30 per cent of plasma and all original Leucocytes and platelets . B.) Centrifuged red cells: They have a PCV of 70- 80 percent, 10-20 percent of plasma and all original Leucocytes and platelets, C.) Red cells with additive (Adsol or SAG-M): They have PCV of 50-60 percent, minimum plasma and all Leucocytes and platelets.

32. Differences Sedimented red cells They have a PCV of 60-70 percent, 30 per cent of plasma and all original Leucocytes and platelets . Centrifuged red cells They have a PCV of 70- 80 percent, 10-20 percent of plasma and all original Leucocytes and platelets,

33. Platelet Concentrate.

34. Types of Platelet Preparations Preparation for platelet transfusion starts from the production of quality-approved platelet concentrates (PC) in the blood banks. PC can be prepared from whole blood or by apheresis. The shelf life of a PC is five days, within which it must be used. The normal dose of platelet transfused is calculated as 10 to 15 ml/kg of the patient. A transfusion rate of 2 to 5 ml/min is used, thereby completing the transfusion in 1 to 2 hours. Slower flow rates are used in patients at risk of fluid overload .  6 whole blood unit-derived platelets equal one apheresis platelet, which contains 5X10^10 platelets per unit.

35. Preparation of Platelets Platelet concentrates are usually prepared from whole blood either through Buffy coat method or as PRP

36. Component Separation

37. Preparation of Platelets by PRP 1. Whole blood units intended for platelet preparation were placed in diagonally opposite cups of refrigerated centrifuge and temperature set at 20–24°C . 2. Then these blood bags centrifuged , - light spin (1300 rpm for 13 minutes). 3. The supernatant PRP was transferred to a satellite bag and red cells transferred the optimal additive solution (saline, adenine, glucose, mannitol; SAG-M) to the primary bag containing red cells. A dielectric sealer was used to seal the tubing between the primary bag and the satellite bag. 4. After proper labeling, the separate primary bag containing the red cells was then stored at 2–6°C. 5 . Then the PRP was processed to yield concentrate of platelets.

38. Preparation of platelet concentrate from PRP Preparation of platelets from PRP begins with : 1. A soft spin (1300 ) for 5 minutes of the WB, at room temperature ( 22-34 C), the RBCs settled to bottom, plasma reached platelet collected in separate bag. 2 . Bags containing PRP were then centrifuged for 15 minutes at 20–24°C at 3000 rpm (heavy spin). 3. After separating platelet-rich plasma (PRP) from the red cells, the PRP is centrifuged at a heavy spin for a longer time. This time platelets settle to the bottom of the bag. 4. The plasma (platelet poor) is transferred into another satellite bag. 5. Platelet pellet obtained, is held undisturbed for 30 to 60 minutes. At the end of one hour, the platelets in the resident autologous plasma were evenly resuspended by hand manipulating the platelet storage bags. 6. Once platelets are resuspended, they are stored at room temperature (between 20 to 24 C) with continuous agitation during storage.

39. Separation of Platelet from WB.

40. PLATELET CONCENTRATE ( BUFFY COAT METHOD ): Contrast to the PRP preparation method, the first step in buffy coat method is high g-force centrifugation followed by a low g-force step. 1. A buffy coat layer is obtained between the red cells and the plasma which appear after primary centrifugation. 2.The red cell and the plasma are transferred to their respective bags and sealed. 3.The satellite bag containing the buffy coat along with some plasma and red cells is the hanged and allowed to rest. 4. Followed by a soft spin centrifugation , the supernatant platelet is separated and drawn off in a platelet bag and the remaining buffy coat is discarded after separation. 5.The buffy coat method yields more plasma, greater red cell loss, better initial white blood cell (WBC) reduction before filtration, and moderate reduction in viable bacteria in the platelets.

41. BUFFY COAT METHOD Compared to the PRP preparation method, the first step in buffy coat method is high g-force centrifugation followed by a low g-force step. A buffy coat layer is obtained between the red cells and the plasma which appear after primary centrifugation . ↓ The red cell and the plasma are transferred to their respective bags and sealed . ↓ The satellite bag containing the buffy coat along with some plasma and red cells is the hanged and allowed to rest . ↓ Followed by a soft spin centrifugation, the supernatant platelet is separated and drawn off in a platelet bag and the remaining buffy coat is discarded after separation . (The buffy coat method yields more plasma, greater red cell loss, better initial white blood cell (WBC) reduction before filtration, and moderate reduction in viable bacteria in the platelets).

42. Apheresis Collection of platelets from a donor by special technique called Apheresis. Here the blood drawn from a donor spins inside the tubes of the machine, separates in to three layers, the heavier red cells settles to the bottom, plasma at the top and platelets in between, the platelets tracked and collected in a separate bag, RBCs transfused back. One can donate 2 to 3 doses of platelets in a single visit.   One can donate platelets every 7 days up to 24 times a year.

43. Calibration of refrigerated centrifuge and platelet incubator and agitator 1. For optimum speeds and spin times for platelet separation, the refrigerated centrifuge was calibrated. 2. The accuracy of the digital thermometer was verified annually in addition to the daily temperature check. 3.The accuracy of speed and time was checked by the hospital’s biomedical engineering department for a period of six months with an accurate tachometer and stop watch. 4.The optimal spin times and speed were also evaluated in conjunction with the evaluation of platelet concentrate QC by function checks. 5. Regularly calibrated platelet incubators designed to provide a controlled environment for stored platelets and also to accommodate platelet rotators to ensure optimum recovery and function of stored platelets. 6. Temperature recorder charts have been maintained and regularly replaced to ensure the instrument’s temperature range. 7.Also performed were periodic preventive maintenance and cleaning works as well as regular inspection of sensor bottle fluid.

44. Quality testing of platelet concentrates . The quality of platelets during storage can be evaluated by determining the recovery and survival of the transfused platelets in thrombocytopaenic patients. A 1 h post-transfusion CCI of 10–20,000 is considered a good response while a CCI of less than 7500 is a poor response and indicates transfusion.

45. Quality assessment of platelet concentrates 1. Total platelet count of the unit.( Total number of platelets/unit of platelet concentrate = platelet count/ μL of the platelet concentrate × 103 × volume of residual plasma present in the unit . ) 2. Measurement of pH of platelet concentrate unit.( This method of pH measurement permitted the differentiation of pH values within the range of 5–9 . ) 3. Measurement of volume of platelet concentrate unit.( The volume was indirectly calculated by weighing the blood bag using a digital weighing scale. ) 4. Visual inspection of platelet concentrate unit. 5. Residual leukocyte and RBC contamination of platelet concentrates

46. Visual inspection of platelet concentrate unit It was performed to detect the presence of excessive platelet aggregation and contamination. Units with excessive platelet aggregates were discarded. Pink or red discoloration of the units was a measure of red cell contamination and such units were issued only after performing the cross-matching tests in the unit. Also review the “swirling” or “shimmering” appearance of platelet by holding the bag against a good light source and squeezing it gently.

47. Leukoreduction Leukocyte reduction is a method to reduce the leucocytes in the collected blood before it is transfused to the patient. It can be done before storage in the blood bank or before transfusion at the bedside. Disadvantages of Leucocytes present in the blood. Non hemolytic transfusion reactions ( febrile reaction). Immunization of recipient to HLA or granulocyte antigens. CMV lives in granulocytes. Transmission of Leucotropic viruses Transfusion Associated Graft Versus Host Disease (TAGVHD) Reaction to cytokines produced by granulocytes . Transfusion Related Acute Lung Injury (TRALI) Transfusion related Immuno suppression.

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