Blood component – Principles of separation & indication.pptx

Transfusion Medicine

1. Blood components - Principles of separation & indications Dr. Topon Narzary, MD (Path) Assistant Professor, Pathology Department Diphu Medical College & Hospital, Diphu (Assam) Department of Pathology, Diphu Medical College & Hospital

2. Introduction “Blood is the best substitute of lost blood ” Effective blood transfusion therapy depends on availability of different blood components Components when used separately or in combination can meet most patients transfusion needs and keep the risk of transfusion to minimum.

3. History 1628 : William Harvey introduces the controversial concept of circulation. 1667 : Jean Baptist Denis in France and Richard Lower in England separately report giving the first human blood transfusion with blood from lambs. 1818 : James Blundell performs the first successful transfusion of human blood to a patient. 1914 : Sodium citrate is found to prevent blood from clotting, allowing blood to be stored between collection and transfusion. 1936 : Chicago’s Cook County Hospital establishes the first true “blood bank” in the United States. 1950-1960 : Component therapy started.

4. Why Component separation is justified ?

5. Separation of blood components allows optimal survival for each component. Allows transfusing specific blood components according to the need of the patient Avoids use of unnecessary component which may be contraindicated in a patient. Several patients can be treated from one unit of donated blood. Use of blood components supplements blood supply and adds to the blood inventory.

6. Why whole blood is not rational ? Maximize blood resource : In addition to what patient requires, there is unnecessary administration of unwanted cell or plasma constituents Whole blood – 1 patient Component therapy – 4 patients Packed red cells – thalassemia Plasma - liver d/s, burns Platelets - thrombocytopenia cryopecipitate - hemophilia

7. Whole blood Whole blood contains 350 ml of donor blood plus anti-coagulant solution(CPDA) of 49 ml. Hematocrit of 30 to 40 %. Minimum 70% of transfused RBC survive in recipient circulation 24 hrs after transfusion. Has no functional platelets or labile coagulation factors V &VIII after 48 hrs. Raises Hb by 1 g/dl or hct by 3% Indication : Acute massive blood loss, exchange transfusion .

8. Various Blood components CELLULAR COMPONENTS Red cell concentrate Platelet Concentrate Platelet Apheresis Granulocytes Apheresis PLASMA COMPONENTS Fresh frozen plasma Single donor plasma Cryoprecipitate Cryo -poor plasma PLASMA DERIVATIVES Albumin 5% & 20% Plasma Protein Fractions Factor VIII concentrate Immunoglobulins Fibrinogen Other coagulation factors

9. Preparation of Blood components is possible due to : Muliple plastic packs system Refrigerated centrifuge. Different sp. Gravity of cellular components:- Red cells specific gravity 1.08 - 1.09 Platelet specific gravity 1.03 - 1.04 Plasma specific gravity 1.02 - 1.03 PRINCIPLE : Differential centrifugation technique

10. Mathematical formulae RPM Formula : RPM = √[ RCF/(r x 1.118)] x 1 x 10 5 RCF (g Force) = Relative Centrifugal Force r = radius of centrifuge rotor in cm (rotational radius) G Force (RCF) Formula : Relative Centrifugal Force (RCF) in g = (RPM) 2 x 1.118 x 10 -5 x r r = radius of centrifuge rotor in cm (rotational radius)

11. Conversion of centrifugal speed from g to rpm

12. Guidelines Transfusion Medicine T echnical M anual (WHO) NACO (National AIDS Control Organization) American Association of Blood Banks (AABB) Department of Pathology, Diphu Medical College & Hospital

13. Blood Collection Bags Blood collection bags have closed intregral tubing. After separation various component can be transferred from one bag to another in a closed circuit thus maintaining sterility.

14. Interconnected Bag System Double packs system : R ed cells, and Plasma only. Triple packs system : R ed cells, Platelet concentrate and Fresh frozen plasma. Quad packs system : Red cells Platelet concentrate Cryoprecipitate (factor VIII) and Cryo- poor plasma.

15. Precautions to be observed in Component separation PRECAUTIONS DURING COLLECTION OF BLOOD Selection of Donor : weight , age, and Hb status. Clean and aseptic venepuncture to minimise bacterial contamination. Ensure Free flow of blood . If any unit takes more than 8 minutes to draw, it is not suitable for preparation of platelets concentrate, fresh frozen plasma or cryoprecipitate. Correct amount of blood proportionate to anticoagulant should be collected. Platelets should be separated within 6-8 hours from the time of collection of blood.

16. PRECAUTION DURING CENTRIFUGATION Opposing cups with blood bag & satellite bag must be equal in weight. Weight should be used for balancing. Bags should be placed that its broad side faces the outside wall of the cup. Correct speed of centrifugation and time must be maintained .

17. Thematic presentation Whole Blood Processed within 8 hours ) Packed R ed cells (PRBC) Fresh frozen plasma (FFP) Platelets (PLC)

18. Contd... C ryoprecipitate Cryo poor plasma (CPP) Whole Blood Fresh frozen plasma

19. Platelet rich plasma (PRP) Principle - Differential centrifugation Red cells Packed cells Red cells + additive Plasma Bank plasma Fresh frozen Cryo supernate Platelets Platelet rich concentrate Platelet rich plasma Cryoprecipitate Whole blood Red cells

20. Whole blood Light spin 1750 rpm for 9 min at 22 C Packed red cell Platelet rich plasma Heavy spin 3850 rpm at 22 C for 9 min Plasma Platelet concentrate Thawing after freezing at -60 C CRYOPRECIPITATE CRYO-POOR PLASMA

21. Preparation of Red cell concentrates RBC PREPARATIONS ARE:- SEDIMENTED RED CELLS :- PCV of 60 to 70%, 30 % of plasma and all original leucocytes & platelets. CENTRIFUGED RED CELLS :- PCV of 70 to 80%,15% plasma and all original leucocytes & platelets. RED CELLS WITH ADDITIVE( Adsol or SAG-M ):- PCV of 50 to 60%,minimum plasma & all leucocytes & platelets.

22. Red cell in additive solution( adsol,sag -m) Blood is collected in the primary bag of additive system, consisting of a primary bag containing anticoagulant solution CPD attached with at least two satellite bags, one of which is empty and another contains 100 ml of additive solution e.g. Adsol or SAG-M Centrifuged at 1750 rpm for 9 mins Plasma is transferred to the transfer bag and Then SAG-M is transferred to the PRBC and separated. Stored at 2-6 ◦C.

23. ADVANTAGES Maximum amount of plasma can be removed for preparation of plasma components. Shelf life increases from 35 days to 42 days . Flow of infusion is increased due to reduced viscosity. Department of Pathology, Diphu Medical College & Hospital

24. Preparation of Platelet Rich Plasma(PRP)& Platelet concentrate(PLC) Platelet concentrate can be prepared from: 1. Random donor platelet (prepared from 450 ml whole blood) 2. Single donor platelet prepared by apheresis Department of Pathology, Diphu Medical College & Hospital

25. Random Donor Platelet Procedure: 1 . Collect 350 ml blood by a clean, single venepuncture into CPDA or Adsol / SAGM triple bags system. 2. Keep the blood bag at room temperature (20-22°C) before preparing platelets, for not more than 6 hours. 3. Centrifuge the blood bags at 20-24°C at light spin for appropriate time (1750 rpm for 9minutes). 4. Separate 4/5 of the Platelet rich plasma (PRP) into one satellite bag. Double seal the tubing between the primary bag and the satellite bags. Separate the primary bag with red cells. One of the satellite bag contain PRP. PRP may be used as such or processed further to prepare Platelet Concentrate (PC).

26. Schematic diagram

27. 5. Centrifuge the bag with PRP and another satellite bag at 20-24°C at ‘heavy spin’ for appropriate time e.g. 3850 rpm for 9 minutes. 6. Express supernatant platelet - poor plasma into another empty satellite bag. 7. Leave approximately 50 ml of plasma with the platelets and label it. 8. Leave platelet concentrate (PC) undisturbed at 20-22°C for 1 hour, then re-suspend the platelet in plasma by gently mixing for l0 min. 9. Store platelet at 20-22°C under constant agitation in platelet incubator with agitator till used. The shelf life is 3-5 days.

28. Apheresis Apheresis (or hemapheresis ) is a Greek word that means to separate or remove. In apheresis blood is withdrawn from a donor or patient in anticoagulant solution and separated into components. One (or more) component is retained and the remaining constituents are returned to the individual Department of Pathology, Diphu Medical College & Hospital

29. Automatic Apheresis machine

30. PLATELET APHERESIS RDP Prepared from WB Average > 3 x 10 11 platelets(equal to platelets obtained from 5 to 6 whole blood donations) Plasma volume 200 ml Leukocytes < 5.5 x 10 6 in each unit obviate the need of filtration Red cells - Traces pH - 6.0 or more Exposes a patient to one donor Less exposure to infections Low risk to alloimmunization 5.5 x 10 10 platelets 50-60 ml filtration is required to reduce leukocytes more pH - 6.0 or more Exposes a patient to multiple donors More exposure to infections more risk to alloimmunization

31. HLA - or platelet- matched donor product can be prepared for the patients who have become refractory to platelets Decreased risk of bacterial contamination and easy handling as platelets are pooled Donation by apheresis requires great commitment Sophisticated equipment required Highly trained personnel required Not possible More risk of bacterial contamination requires Routine donation can be made from Whole blood Not required Not required

32. Advantages of Apheresis Apheresis technology is commonly used to collect platelets because a full therapeutic dose of platelets (equivalent to six whole blood–derived platelet units) or even as many as three therapeutic doses (equivalent to 18 whole blood–derived units) can be obtained from one apheresis donation Department of Pathology, Diphu Medical College & Hospital

33. Fresh Frozen Plasma (FFP) FFP is prepared by separating citrated plasma from whole blood and freezing it within 8 hours of collection or by freezing citrated apheresis plasma within 6 hours of collection. FFP may be stored at -18°C or below for up to 1 year It contains all coagulation factors and great care must be taken during collection of blood, freezing and thawing to preserve their activity. One unit of FFP is around 225-250 ml

34. Cryoprecipitate Cryoprecipitate is made from one unit of Fresh Frozen Plasma. Cryo is the insoluble portion of plasma that precipitates when a unit of Fresh Frozen Plasma is thawed between 1 - 6° C. The excess plasma is removed from the precipitate, creating Cryoprecipitate Poor Plasma ( Cryo -Poor Plasma.) Stored : -30 C Each unit of this cryoprecipitate contains approximately 80 to 120 units of factor VIII 150 mg of fibrinogen Factor XIII High-molecular-weight multimers of vWF

35. Blood Component Modification

36. Leucocyte reduction Leukocytes in blood components can cause: Non- hemolytic febrile transfusion reaction (NHFTR) Human leukocyte antigen (HLA) alloimmunizaion Transmission of leukotropic viruses Cytomegalovirus(CMV), Epstein- Barr virus (EBV) Human T-cell lymphotropic virus type 1 (HTLV-1) Transfusion related Graft versus host disease Transfusion related acute lung injury (TRALI) Transfusion related immunosuppression

37. Methods of the preparation of Leucocyte -Reduced Red cells Centrifugation and removing of buffy coat Filtration Washing of red cells with saline Freezing and thawing of red cells

38. Washed Products Packed red cell can be washed with normal saline to remove plasma proteins,white cells,and platelets. Washing may also be used to reduce the concentration of potassium in RBC supernatants, which may be required prior to massive or rapid infusion of stored RBC to neonates. Washing is used primarily to prevent severe allergic reactions , which are thought to be triggered by donor plasma proteins.( IgA deficient individual who developed anti- IgA ANTIBODIES

39. Irradiation of Blood Products Gamma-irradiation(25 Gy ) of cellular blood components is used to prevent transfusion-related GVHD by inhibiting replication of donor lymphocytes in the blood component. USED in immunodeficient individual and patient receiving blood from first degree relative.

40. Blood component preparation in our department

41. Plasma and RBC after first centrifuge

42. Packed RBC found after first centrifuge at 1750 RPM Department of Pathology, Diphu Medical College & Hospital

43. Platelet and Plasma after 2 nd centrifuge at 3850 RPM

44. Department of Pathology, Diphu Medical College & Hospital

45. Uses of Blood components Because each whole blood unit constituted approximately 10% of a donor’s blood volume, each component can be considered roughly 10% replacement therapy for an adult patient. Considering one unit Whole blood = 450 ml Department of Pathology, Diphu Medical College & Hospital

46. INDICATIONS OF RED CELLS TRANSFUSION ARE : In decreased bone marrow production conditions Leukemia Aplastic anemia In decreased red cells survival conditions Hemolyic anemia Thalassaemia In bleeding patients Surgical bleeding Traumatic bleeding

47. Leukocytes reduced Red blood cells Multitransfused patients like thalassaemic . Leukemia Aplastic anemia Immunosupressed & Immunodeficient . Multiparous women. Prevention of recurrent FNHTRs Prevention or delay of primary alloimmunization to HLA antigen Prevention of CMV transmission in at risk individual

48. Washed Red cells Washing of red cells removes 70 - 95 % of leukocytes 15 - 20 ml of red blood cells Plasma proteins and microaggregates .  Indications Currently it is mainly used to prevent allergic reactions . Patients having recurrent attacks FNHTRs IgA deficient patient who has developed anti- IgA . Paraoxymal noctural hemglobiuria (PNH), sensitive to complement. Patients who have developed antibodies to plasma proteins.

49. Frozen / Deglycerolized Red cells Polge et al. In 1949 observed that If glycerol ( cryoprotective agent) is added to the cells they can be frozen and thawed without damage for a very long time. Two concentrations are used to glycerolize red cells, high concentration glycerol 40% low concentration glycerol 20% Frozen cells are deglycerolized before transfusion . Removal of glycerol is achieved by washing the RBC with decreasing conc. of saline.

50. Use Frozen red cells are primarily used for autologous transfusion The storage of rare group blood Department of Pathology, Diphu Medical College & Hospital

51. Platelet concentrate Indications of platelet transfusion when   Platelet count is < 5000 / μl regardless of clinical condition Platelet count is 5000-10000/ μL , if there is increased risk of bleeding due to haematological malignancies. Platelet count is 10000-20000/ μL , if thrombocytopenic bleeding is present . Chemotherapy for malignancy, if platelet count is 20000/ μL DIC Massive transfusion. In major surgery , if platelet count is < 70-80000/ μL

52. Granulocyte According to AABB standards, each leukapheresis should yield at least 1 × 10 10 granulocytes Indication : Severe neutrophil depletion Antibiotic resistance Granulocytes should be administered on a daily basis until the patient’s endogenous neutrophil count rises to 0.5 × 10 9 /L or until the infection clears (AABB guideline)

53. Fresh Frozen Plasma : Contents of 1 unit of FFP prepared from 450 ml o f whole blood   Plasma 225 - 250 ml All coagulation Factors 1 i.u. / ml of each factor (including Factors V & VIII) Fibrinogen 200 - 400 mg Department of Pathology, Diphu Medical College & Hospital

54. Indications of Fresh Frozen Plasma Actively bleeding and multiple coagulation factors deficiencies in Liver disease Disseminated intravascular coagulation (D1C) Coagulopathy in massive transfusion TTP When specific disorder cannot be or has not yet been identified Familial Factor V deficiency Congenital or acquired coagulation factor deficiency Antithrombin III deficiency

55. Cryoprecipitate Each bag has approximately : Plasma 10- 15 ml Factor VIII 80 - 100 i.u . Fibrinogen 150 - 250 mg von-Willebrand Factor 40 - 70% Fibronectin 55 mgm Factor XIII 20 - 30% of the original Department of Pathology, Diphu Medical College & Hospital

56. Indications Hemophilia A von Willebrand’s disease Congenital or acquired fibrinogen deficiency Acquired Factor VIII deficiency (e.g. DIC, massive transfusion) Factor XIII deficiency Fibrin glue : Cryoprecipitate has also been used topically, along with thrombin and calcium, as a “fibrin glue.”

57. Commercial Plasma derivatives Albumin: Prepared by ethanol fractionation. Found in 5 % or 25% preparation Solvent/treated – Treated plasma : Prepared from many FFP units treated with organic solvents to make then free from lipid enveloped viruses ( HBV HIV etc) but it doesn’t have any effect on non lipid enveloped viruses (HAV, Parvo virus ) Coagulation factor concentrate : Factor VIII concentrate Intramuscular immunoglobulins : Contains 95% IgG and small amount of IgA and IgM Intravenous immune globulins: IV Rh immune globulin.

58. Conclusion Fully automated machines have revolutionized the component preparation. Collaboration between transfusion medicine professional and clinicians is important. Proper care must be taken to prevent transfusion of infection to the recipient. Proper quality control should be ensured each and every time. Department of Pathology, Diphu Medical College & Hospital

59. Tube sealer CRYOFUGE (CENTRIFUGE)

60. Department of Pathology, Diphu Medical College & Hospital

61. Thank you Department of Pathology, Diphu Medical College & Hospital