Blood component storage and screening.pptx

Blood component storage and screening

1. Blood component storage and screening

2. Blood components Blood components include Whole blood Packed RBC Fresh frozen plasma(FFP) Cryoprecipitate Platelet rich plasma Plasma derivatives

3. COLLECTION OF DONOR BLOOD

4. Blood from a donor is collected in a closed system of sterile, disposable plastic bag (single, double, or triple bag depending on component to be prepared) with 350 mL capacity These bags contain 49 mL of citrate phosphate dextrose adenine (CPDA-1) solution (14 mL solution is required for 100 mL of blood) Currently, CPDA-1 solution is commonly used in which blood can be kept stored at 2-6°C for 35 days Blood bag containing anticoagulant-preservative solution Equipment for blood collection

5. Single bag : Whole blood Double: Red blood cells, plasma Triple: Red blood cells, platelets, cryoprecipitate or plasma Quadruple: Red blood cells, platelets, cryoprecipitate, plasma In India, whole blood transfusion is still practiced at many places, and therefore, single bag is commonly used. Double or triple bag systems are used if facilities for separation of components are available

6. COLLECTION OF BLOOD BY APHERESIS Apheresis is a procedure in which a particular blood component is collected and the remainder of the blood is returned back to the donor by an automated machine Blood is separated into components based on their specific gravity by centrifugal force In a closed system, blood flows from the donor’s arm into the centrifuge bowl, a specific component is removed, and the remaining blood is returned to the donor The procedure may take 30 minutes to 2 hours

7. This technique allows collection of larger volume of a specific component and decreases exposure of the patient to the number of donors Donor apheresis is of two main types: plasmapheresis and cytapheresis Plasmapheresis: Plasma is harvested from the donor and the cellular components are returned to the donor

8. Cytapheresis : Individual cellular components are harvested from whole blood by cell separator machine. Cell components that can be collected include Red cells (Red cell apheresis) Two units can be collected from donors who are larger and have higher hematocrit than for single red cell donations Plateletpheresis This is also known as single donor platelets. Can be safely repeated after 4 weeks The advantage of this procedure is HLA-matched platelets can be collected for patients who are refractory to random donor platelets due to development of antiHLA antibodies ( 1 SDP = 5-6 RDP )

9. Leucapheresis : This is collection of granulocytes by apheresis Stem cell collection : Hematopoietic stem cells can be collected from peripheral blood of donor by apheresis for reconstitution of bone marrow in patients with hematologic malignancies, aplastic anemia, and hemoglobinopathies

10. Processing of donor blood ABO and Rh grouping Screening and identification of unexpected antibodies Screening tests for infections transmissible by transfusion – Hepatitis B surface antigen Test for antibodies to hepatitis C virus Test for antibodies to human immunodeficiency virus – Venereal Disease Research Laboratory (VDRL) test for syphilis Blood smear for malaria parasite

11. Red cell storage lesion Definition : Decreased red cell viability and function associated with certain biochemical changes that occur when blood is stored at 4-6 o C Decreased gluose , ATP, pH and 2,3 BPG Increased lactic acid and plasma potassium Changes in red cell shape from biconcave to echinocyte Progressive loss of viability of red cells Shift of oxygen dissociation curve to left

12. Changes occuring during storage Loss of viability of red cells Viability refers to the capacity of red blood cells to survive in recipient’s circulation after transfusion There is progressive loss of viability with increasing duration of storage Storage conditions should be such that, after transfusion, at least 75% of transfused red cells should survive at 24 hours in the recipient’s circulation Shelf-life of the stored whole blood is based on this criterion For whole blood stored in CPDA-1 and maintained at 2° to 6°C, shelf-life is 35 days

13. Depletion of ATP Progressive loss of ATP with increasing length of storage causes decreased deformability of red cells and impairment of Na+/K-ATPase pump Reduction of 2, 3-diphosphoglycerate (2,3-DPG) Progressive depletion of 2, 3-DPG with storage increases oxygen affinity of hemoglobin and reduces release of oxygen to the tissues

14. Formation of microaggregates In stored blood, aggregates of aged platelets, leukocytes, and cold insoluble globulin form Their number and size increase with increasing length of storage Loss of granulocyte function Loss of granulocyte function (phagocytic and bactericidal property) occurs within 24 hours and loss of platelet function occurs within 48 hours of blood collection

15. PRESERVATION AND STORAGE The ability of transfused RBCs to deliver oxygen to tissues and survive in the patient’s circulation is the best measure of how well the RBCs were preserved and stored Survival of transfused RBCs is acceptable when at least 75% of the transfused RBCs are present in the circulation for 24 hours The development of blood preservatives provided a means to store RBCs to facilitate blood banking with minimally toxic anticoagulants

16. Storage solutions CPDA-1 (Citrate Phosphate Dextrose Adenine) – Most commonly used Shelf life 35 days at 2-6 degrees Acid citrate dextrose (ACD) Shelf life 21 days at 2-6 degrees Citrate phosphate dextrose(CPD) Shelf life 21 days at 2-6 degrees

17. CPDA-1 Contents : Trisodium citrate 26.3 g/L Citric acid 3.27 g/L Monobasic sodium phosphate 2.22 g/L Dextrose 31.9 g/L, adenine 0.275 g/L Citrate prevents coagulation by chelating calcium Phosphate acts as a buffer by minimizing decrease in pH that preserves 2,3-DPG better Dextrose (glucose) supports ATP generation by glycolysis Adenine acts as a substrate for red cell ATP synthesis

18. Additive and Rejuvenation Solutions Adsol consists of adenine (to help maintain ATP during storage), dextrose, saline, and mannitol SAGM- Saline adenine glucose mannitol It contains 60% more adenine and approximately 2.5 times as much glucose as CPDA-1 The addition of mannitol prevents excessive hemolysis over the storage period Shelf life increases from 35 to 42 days with additive solution Adsol and SAGM

19. C- citrate – Anticoagulation P- Phospahte – Maintain pH D-Dextrose and A-Adenine – to maintain ATP SAGM or Adsol – higher adenine for ATP and mannitol to prevent hemolysis

20. Blood components

21. Whole blood A single whole blood donation can be separated into different components to provide treatment to more than one patient One unit of whole blood can be broken down into one unit of packed red cells, one unit of platelets and one unit of fresh frozen plasma/cryoprecipitate This process avoids wastage of collected whole blood (each component is stored at a temperature that is optimal for that component) and allows administration of specific replacement therapy

22. Whole blood is centrifuged at light spin to obtain platelet-rich plasma (PRP) PRP is pushed through the attached tubing into an empty satellite bag, while red cells remain in the original bag The satellite bag containing PRP is centrifuged again at a heavy spin 50–70 ml plasma is removed from platelets by expressing it into another empty satellite bag and the platelet bag is sealed The plasma-containing bag can be processed further to prepare fresh frozen plasma and cryoprecipitate

23. Whole blood Whole blood is one unit of unmodified donor blood collected in a suitable anticoagulant-preservative solution (CPDA-1) Its total volume is about 400 mL (350 mL of blood + 49 mL of anticoagulant) It consists of cellular elements (red cells, white cells, and platelets) and plasma Whole blood is stored in an approved blood bank refrigerator at 2–6°C (Shelf life 35 days)

24. It does not contain functionally effective platelets and labile coagulation factors (F V and F VIII) It can cause circulatory overload in patients who require only packed red cells to improve oxygen-carrying capacity Whole blood must be ABO-identical to the recipient

25. Indications Acute blood loss with hypovolemia Exchange transfusion in neonates Nonavailability of red cell concentrate or suspension Contraindication Chronic anemia with compromised cardiovascular function

26. Red cell components

27. Packed red cells Packed red cells are prepared by removing most of the plasma from one unit of whole blood (hematocrit 70–75%) Whole blood is either allowed to sediment overnight in a refrigerator at 2–6°C or is spun in a refrigerated centrifuge Supernatant plasma is then separated from red cells in a closed system by transferring it to the attached empty satellite bag

28. Packed red cells Indications Anemia: Chronic severe anemia, severe anemia with congestive cardiac failure, anemia in elderly Acute blood loss (transfused along with a crystalloid or a colloid solution)

29. Red cells in additive solution (Red cell suspension) These are red cells with minimal residual plasma and an additive solution After collection of whole blood in CPD and separation of plasma, additive solution is transferred from the attached satellite bag/pouch to packed red cells within 72 hours of collection Enhance shelf-life of packed red cells up to 42 days Examples : AS-1 and AS-5: Saline, adenine, dextrose, mannitol AS-3: Saline, adenine, dextrose, sodium citrate

30. Leukocyte-depleted red cells Leucocyte-poor red cells contain < 5×10 6 white cells per bag Methods for leucocyte depletion are : (1) Leucocyte-reduction filters during infusion (2) Filtration during component preparation Indications for leukocyte-poor red cells are : (1) Prevention of HLA immunization in patients who are likely to receive allogeneic bone marrow transplantation (2) Prevention of febrile nonhemolytic transfusion reactions in persons receiving multiple transfusions (3) Prevention of transmission of cytomegalovirus, as this virus resides within the cytoplasm of leucocytes

31. Washed red cells Red cells can be washed with normal saline to remove plasma proteins, white cells, and platelets Such red cells are used for IgA-deficient individuals who have developed anti-IgA antibodies, as exposure will lead to anaphylaxis Shelf-life of red cells after washing is 24 hours at 4–6°C About 20% of red cells are lost in the process

32. Frozen red cells If a cryoprotective agent such as glycerol is added, red cells can be stored frozen for up to 10 years when stored at or below –65° Glycerol prevents red cell dehydration and formation of ice crystals, which causes red cell lysis After deglycerolization (washing with decreasing concentrations of saline), red cells can be stored at 4–6°C for 24 hours

33. With this method, virtually all plasma, anticoagulant, leucocytes, and platelets are removed This method can be used for storage of donor red cells with rare blood groups, for future autologous transfusion, and for individuals who have repeated febrile nonhemolytic transfusion reactions

34. Irradiated red cells Viable T lymphocytes in donor blood can induce fatal transfusion-associated graft-versus-host disease in at-risk patients Gamma-irradiation (2500 cGy) of red cells inactivates T lymphocytes and prevents graft vs. host disease This component can be stored at 4–6°C up to the original expiry date or 28 days from irradiation, whichever is earlier

35. Irradiated red cells Indication Intrauterine or premature neonate transfusions Individuals with immunodeficiency In those receiving blood from first-degree relative donors During stem cell transplantation Patients on immunosuppressive chemotherapy regimens

36. Summary of pRBC characteristics and indications Product Manipulation/Comment Indications Whole blood None Trauma, Massive surgical bleeding pRBC Volume reduced Symptomatic anemia , Hemorrhagic shock pRBC , Leukoreduced White blood cell reduction Prevent transfusion reactions Reduce alloimmunization Reduce infectious diseases Minimize immunomodulation pRBCs , washed Plasma removal Recurrent severe allergic reactions IgA deficiency with anti-IgA pRBCs , irradiated Inactivation of lymphocytes Prevention of Transfusion Associated-GVHD pRBC frozen deglyceralized Frozen with subsequent deglycerolization Facilitates long-term storage Storage of RBC rare phenotypes Autologous storage

37. Platelet concentrate Platelet concentrates can be obtained from whole blood or by plateletpheresis Random donor platelets are prepared from whole blood unit If platelets are to be prepared from whole blood, the unit should be kept at room temperature and the platelets must be separated from whole blood within 6 hours of collection

38. RDP – Random donor platelets One unit of whole blood is centrifuged (light spin) to obtain platelet-rich plasma (PRP) PRP is then transferred to the attached satellite bag and spun (high spin) to get platelets at the bottom and supernatant plasma Most of the supernatant is returned back to the primary collection bag or to another satellite bag, leaving behind 50–60 mL of plasma with the platelets

39. Platelets are stored at 20–24°C with continuous agitation (in a storage device called platelet agitator) Maximum period of storage is 5 days One unit of platelet concentrate contains minimum of 5.5 × 10 10 platelets Transfusion of one unit will raise the platelet count in the recipient by about 5,000/ L Usual adult dose : 4–6 units of platelet concentrate (or 1 unit/10 kg of body weight)

40. Single donor platelets In plateletpheresis, a donor is connected to a blood cell separator machine in which whole blood is collected in an anticoagulant solution, platelets are separated and retained, and remaining components are returned back to the donor With this method, a large number of platelets can be obtained from a single donor (equivalent to 6 units of platelet concentrate or >3.0 × 10 11 ) and exposes recipient to fewer donors This method is especially suitable if HLA-matched platelets are required

41. Indications of platelet transfusion Bleeding due to decreased platelet production Bleeding in hereditary disorders of platelet function Massive blood transfusion Contraindication Thrombotic thrombocytopenic purpura Hemolytic uremic syndrome

42. PLASMA COMPONENTS FFP is prepared from whole blood within 8 hours of collection because after this time labile coagulation factors are lost Plasma is separated from whole blood by centrifugation, expressed into the attached satellite bag, and rapidly frozen at –18°C or at lower temperature FFP contains all the coagulation factors, including labile coagulation factors (F V and F VIII) Fresh frozen plasma (FFP)

43. FFP can be stored for 1 year if temperature is maintained at or below –18°C When required for transfusion, FFP is thawed between 30–37°C (for about 30–45 minutes), and then stored in the refrigerator at 2–6° Since labile coagulation factors rapidly deteriorate, FFP should be transfused immediately or stored for a maximum of 24 hours at 2–6°C Dose of FFP for coagulation factor replacement is 10–20 mL/kg (3–6 units in an adult)

44. FFP Indications Multiple coagulation factor deficiencies: liver disease, warfarin overdose, massive blood transfusion Disseminated intravascular coagulation Inherited deficiency of a coagulation factor for which no specific replacement therapy is available Thrombotic thrombocytopenic purpura/ Hemolytic uremic syndrome (FFP can be used as a replacement fluid for therapeutic plasma exchange)

45. Plasma separated and frozen at –18°C between 8 and 24 hours of collection is called as “Plasma frozen within 24 hours after phlebotomy” It contains all the coagulation factors, but may have reduced levels of F V and F VIII as compared to FFP Plasma frozen within 24 hours after phlebotomy

46. Cryoprecipitate Cryoprecipitate is prepared from plasma that has been freshly separated (within 6 hours of collection) by rapidly freezing it at –18°C or lower and thawing it slowly at 4–6°C The main constituents of cryoprecipitate are High molecular-weight proteins fibrinogen Factor VIII Von Willebrand factor ( vWF ) Factor XIII Fibronectin

47. When needed, cryoprecipitate is thawed at 30–37°C, required donations are pooled and transfused to the patient After thawing, cryoprecipitate can be stored at 2–6°C for 6 hour According to guidelines, each unit of cryoprecipitate should contain >150 mg fibrinogen and >80 IU of F VIII Main indications for cryoprecipitate are deficiency of fibrinogen and dficiency of F XIII

48. Plasma derivatives Plasma derivatives are manufactured by fractionation of large volumes of pooled human plasma After fractionation, derivatives undergo virus-inactivation procedures Plasma derivatives include Human albumin solution F VIII concentrate F IX concentrate Prothrombin complex concentrate Immunoglobulins

49. Transfusion-transmitted infection (TTI) A number of diseases have the potential to be transmitted by transfusion of blood or its components Despite strict donor selection infections can still be transmitted because of inadequate donor screening or due to the donor being in the incubation or ‘window period’ before testing becomes positive for infective markers

50. Viruses HBV 2–6 month incubation period; carrier state; readily transmissible by blood HCV Majority of cases asymptomatic; carrier state; readily transmissible by blood HIV-1 and HIV-2 Carrier state and latent in WBCs; readily transmissible by blood HTLV-I and HTLV-II Latent in WBCs HAV, HEV

51. Cytomegalovirus(CMV) Epstein Barr virus(EBV) Dengue virus West Nile virus

52. Bacteria Treponema pallidum(Syphilis) However, inactivated by storage at 4 ◦C, Parasites Malaria Toxoplasma gondi (Risk only in immunosuppressed patients receiving granulocytes) Chagas disease (Trypanosoma cruzi) Leishmania donovani Babesia microti Prion disease

53. Hepatitis B In India, prevalence of HBsAg carriers is reported to be 1.5 to 4% HBV is highly infectious and is transmitted through all blood components and most of the blood derivatives According to one study in India, incidence of post-transfusion hepatitis following a single transfusion was 6.7% The serological marker first to appear in HBV infection is HBsAg (as early as 5 days after infection)

54. Screening of all blood donations for HbsAg has greatly reduced the risk of transmission of HBV through transfusion. Tests for screening donor blood for HbsAg are Reverse passive hemagglutination assay (RPHA) Enzyme-linked immunosorbent assay (ELISA) Radioimmunoassay (RIA)

55. Reverse passive hemagglutination assay In RPHA, red cells that are coated with anti-HBs antibody are added to the donor’s serum If HbsAg is present in the serum, agglutination of red cells will occur Absence of agglutination indicates negative test The test is called as ‘ passive reverse agglutination ’ because antibody, and not antigen, is artifcially coated on to the red cells

56. Enzyme-linked immunosorbent assay Serum sample to be tested is incubated with anti-HbsAg antibody which has been coated to microtitre plate Test serum is incubated in the well during which binding of antigen from serum (if present) and antibody (attached to the well) occurs An enzyme-linked anti-HBs antibody (conjugate) is added which will combine with the bound antigen A chromogenic enzyme substrate is finally added and the mixture is incubated in the dark

57. If enzyme is present (bound to the antigen), then its action on the substrate will lead to the color development A stopping solution (an acid) is added to prevent any further reaction between the enzyme and the substrate The result is read in a spectrophotometer at the specified wavelength This assay is also called as sandwich assay because antigen is sandwiched between two antibodies

58. Hepatitis C In India, prevalence of HCV is reported to be 1.66% As the amount of HCV antigen in blood stream is small and cannot be detected readily, screening of donor blood for HCV infection is done by detection of anti-HCV antibody in serum (becomes detectable after 6–8 weeks of infection)

59. Earlier tests (first generation ELISA) used recombinant proteins complementary to the NS4 region of the HCV genome Second generation ELISA incorporated recombinant or synthetic antigens from NS4 as well as NS3 regions (c100- 3, c22-3, c33c) of the genome, resulting in improvement in sensitivity and specificity Third generation ELISA, in addition to NS3 and NS4, also includes antigens from NS5 region

60. Human Immunodeficiency Virus Adult prevalence of HIV infection is 0.7%, 90% of which are in the age group of 15–45 years Following HIV infection, viremia becomes detectable after a few days and lasts for several weeks Anti-HIV antibodies appear 6–12 weeks after infection Window period is the period between the onset of HIV infection and appearance of detectable anti HIV antibodies in serum; it is the infectious but seronegative period Transfusion of donor blood collected during the window period will transmit HIV to the recipient

61. HIV screening tests necessary for whole blood donors are: Anti-HIV antibodies (HIV-1 and HIV-2) by ELISA HIV-1 nucleic acid amplification testing (NAT)

62. Nucleic Acid Testing Nucleic acid testing (NAT) is rapidly becoming a standard method for detection of pathogens before the appearance of antibodies It narrows the window period thereby increasing the blood safety It has been reported that with NAT, window for HIV has been shortened to 11 days as compared to 22 days with antibody testing, and window for HCV has been shortened to 10 days from 82 days as compared to antibody testing

63. In this technique, nucleic acid is extracted from donor plasma, amplified, and the viral genetic sequence is identified Very low amounts of viral copies can be detected before the appearance of antibodies HIV NAT testing can be done by two methods: Polymerase chain reaction, and Transcription-mediated amplification

64. Parasites Plasmodium species Malaria parasite can be transmitted through all blood components For detection of malaria parasite, commonly blood smears are examined by light microscopy However, the test is positive only when parasites are >100/  L in blood Rapid diagnostic tests (card tests) that detect parasite proteins are also being used where expertise for microscopy is not available or is operationally difficult

65. Mandatory infectious disease testing HIV- Anti HIV1 and HIV 2 antibodies HBV- HBsAg HCV- Anti HCV antibodies Malaria- Blood smear Syphilis – VDRL test, Rapid plasma regain test *NAT is not currently mandatory in India

66. References Blood banking and transfusion medicine by Hillyer, Second edition Postgraduate hematology by A victor Hoffbrand , Seventh edition Kawathalkar , Essentials of clinical pathology, Third edition John R Rees, Conventional blood banking and blood component storage regulation: opportunities for improvement, Blood transfusion 2010 National Blood transfusion council, Storage and transportation of blood and its components

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