A Very Basic Approach to some of Transfusion Medicine

1. A Very Basic Approach to some of Transfusion Medicine Christina Lee R5 Haematology

2. What are the blood products we use?

3. The comprehensive list: • Packed red blood cells (pRBC) • Platelets • Frozen Plasma • Cryoprecipitate • Plasma Derivatives: • Albumin • Intravenous immune globulin (IVIG) • Factor concentrates (Factor VIII & IV)

4. Types of Blood Products • Generally collected as whole blood (450-500 mls). • One person can donate this amount ~ 3 x year. • However, can collect via apheresis. • This is particularly relevant for platelets since it is at highest risk for bacterial contamination • Also it is a pooled product (~6 donors in 1 unit), which increases risk of transmitted disease, as well as allo-immunization.

5. What is Allo-immunization?

6. Allo-immunization - Definition • Alloimmunization • an immune response generated in an individual by an alloantigen from a different individual • Alloantigen • an antigen existing in alternative (allelic) forms in a species, thus inducing an immune response when one form is transferred to members of the species who lack it; typical alloantigens are the blood group antigens

7. Blood Component Preparation Anticoagulant used is citrate based.

8. Platelets • Platelets remain viable up to 7 days. • Bacterial contamination unacceptably high after 5 days. • Will contain some RBC and plasma. • Pool platelets immediately before transfusion. • Anywhere between 4 to 10 units. • 6 units is approximately 1 unit of apheresis platelets.

9. Frozen Plasma (in Canada its never “Fresh”) • This is the “platelet poor plasma” • Contains all clotting factors including fibrinogen • Stored at -18°C • Shelf life of 12 months • “FFP” – by definition is frozen within 8 hours of phlebotomy • Ensures preservation of labile coagulation factors (V, VIII) • In Canada we have “Frozen Plasma” which by definition is frozen within 24hrs • Because all Canadian products are leuko-reduced. • There is a variable reduction in amount of labile factors. • However, after 48 hours of storage still have 50-76% of factor VIII, and > 75% of factor V.

10. Cryoprecipitate • Concentrated form of fibrinogen and von Willebrand. • FFP is frozen x 24 hours and then thawed in the fridge. • When FFP is in its “slush” phase it is spun at 4°C. • Supernatant plasma is removed leaving cryo in 5-15mL of plasma. • Cryo is then frozen and stored at -18°C for up to 12 months. • Cryo and FFP are also pooled products. • Once thawed both are good for up to 24 hours in the fridge, 4 hours at room temperature.

11. Leukocyte Reduction(All Canadian Products) • Why are transfused WBC bad? • Immunologically-mediated effects. • Consequence of allosensitization to HLA. • Febrile nonhemolytic transfusion reactions. • Platelet refractoriness. • Transplant rejection. • Graft-versus-host disease. • Immunosuppression, reactivation of viral disease. • Infectious disease transmission. • CMV • EBV • HTLV1 • Bacteria (in particular Yersinia and enterocolitica) • Reperfusion injury. • Relates to reperfusion of ischemic myocardium which is known to lead to ultrastructural damage, WBC are thought to play a central role. • Leukoreduction may be an effective way of reducing reperfusion injury after CABG.

12. Leukoreduction • Products are simply filtered. • Can occur pre or post storage. • Pre-storage: • Filtered shortly after processing. • Advantages: • Immediate availability. • Product consistency. • Less transfusion reactions - less cytokines and histamine. • Less alloimmunization, immunosupression & septic transfusion reactions.

13. So what happens when you order a unit of blood?

14. Pre-transfusion Compatibility Testing(First you need to understand what you are testing for.)

15. The Carbohydrate System • Carb. Epitopes on proteins and membrane lipids. • Posttranslational modifications under control of several enzyme known as glycosyltransferases. • Not red cell specific. • Therefore also play roles in organ tranplantation, cell development, cancer, and infectious disease. • Usually naturally occurring.

16. ABO Antibodies • The most clinically relevant! • Cause acute hemolytic transfusion reaction (HTR), Hemolytic Disease of the Fetus and Newborn (HDFN) • The alloantibodies made in the patient can be IgG or IgM • Naturally occurring: • Immune stimulation by transfusion or pregnancy not needed. • Stimulus probably exposure to environmental bacteria (ex normal intestinal flora). • Therefore newborns acquire Abs at ~ 3-6 months of age, achieving adult levels by 5-10 years.

17. Why is this system particularly dangerous? • Cause Intravascular Hemolysis which is BAD! • Bind RBC at 37 C and fix complement. • Transfuse incompatible red cells (“major incompatible”) or plasma (“minor incompatible”) can lead to severe HTR. • HDFN – IgG of Mum (usually O) crosses placenta and hemolyses fetus’ RBC (Fetus is usually A).

18. The Polypeptide System • Polypeptide blood group antigens that reside on endogenous, intrinsic red cell proteins that have roles in red cell maturation and physiology. • Membrane transporters (Rh, Kidd, Colton, Diego, Gil). • Associated with complement and complement regulation (Chido, Knops, Cromer). • Adhesion molecules (Lutheran,LW,Indian,Scianna Ok). • Antibodies are the result of immune stimulation.

19. The Rh System • Phenotypes: • Comprises more than 50 antigens. • Only some are clinically relevant. • 5 antigens present on 2 Rh proteins. • D • Most clinically relevant. • What makes you Rh positive. • Causes HTR and HDFN. • C/c and E/e

20. Rh Antibodies • Alloantibodies against Rh antigens are always clinically significant – HTR & HDFN. • Clear incompatible red cells via extravascular hemolysis. • Require immune stimulation by Tx or pregnancy. • Rh- Mums given Rh Immune Globulin prophylaxis midpregnancy and within 72hrs of deliver. • In the lab they are reactive at 37 C or via IAT. • Immunogenicity - D > c > E > C > e

21. Compatibility Testing – An Intro • The antigenic substances on the surface of red cells that may be recognized as foreign if transfused into a recipient who is not antigenetically identical to the donor. • The antibodies those substances stimulate and then react with. I.E. the alloantibodies that are formed against that antigen (ie not autoantibodies).

22. The Alloantibodies • 1st – not all Ag are equally immunogenic • 2nd – some people tend to be “antibody responders” • 3rd - Different antigens elicit different antibody responses, i.e. IgG or IgM. • Reaction characteristic of the Ab compatibility testing. • Nature of the clinical antibody response. • Structure dictates function.

23. Overview of the Type, Screen & Cross Match

24. The Forward & Reverse ABO Typing Forward: Tests recipients red cells. Reverse: Tests recipients serum to find out what Ab are pesent.

25. Grading Red Cell Agglutination(The Immediate Spin Technique)

26. What This Looks like in Real Life

27. Rh Typing • Rh+ refers to being D+. • Rh- refers to being D-. • Just a forward typing using commercial anti-D. • No reverse typing done since only Rh- patients who have been exposed to Rh-positive blood will be positive.

28. The Antibody Detection TestChecking for alloantibodies in patients serum directed against non-ABO blood group antigens on donor RBC. • Patients plasma tested using a panel of 2-3 group O red cells which are of known antigenic composition. • Really this is an indirect coomb’s test. • AKA “indirect antiglobulin test” • The equivalent methadology is used for: • Antibody identification testing. • Antigen typing. • The full crossmatch.

29. Antibody Detection Test – The Process

30. The Antibody Detection Test was Positive! Identification of the Antibody

31. Antibody Identification – The Panel • If agglutination, hemolysis occur you must identify the Ab so that you can give RBC which are negative for the antigen the alloantibody recognizes. • You perform the indirect antiglobulin test using O RBS which contain a known antigenic composition.

32. Component Selection

33. The Crossmatch – The FINAL Step • Test the patient’s serum with the donors RBC. • The abbreviated crossmatch - an immediate spin technique. • Full cross match requires all 3 phases: • Immediate spin, 37 C incubation, Antiglobulin phase. • Done when recipient has a known antibody. • Will detect any unexpected antibodies not included on the commercial reagents. • If you have an autoantibody, may always be positive.

34. The In-Vivo Crossmatch • There is a protocol for each MUHC blood bank. • Infusion 20 to 30 mL of RBCs from the selected unit of blood. • For 30 minutes, observe the patient for signs and symptoms of a hemolytic transfusion reaction. • At the 30min mark obtain a blood which is examined for the presence of hemoglobinemia (i.e. screening for intravascular hemolysis of the infused cells)

35. Transfusion Reactions What are the different types?

36. What is the general management approach?

37. STOP the Transfusion

38. Order blood work to assess for hemolytic reaction. • Then return/send the following to Transfusion Services: • The implicated unit with infusion set (minus needle). • To track clerical error • For further testing • Culture, gram stain • If hemolysis suspected repeat typing and antibody screening of the unit, repeat crossmatch with posttransfusion serum • A red top (clot tube) or purple top (EDTA tube) containing a sample drawn from the recipient following discontinuation of the transfusion. • Look for hemolysis • DAT • Repeat ABO and Rh typing on pre & posttransfusion samples and on the unit if hemolysis suspected. • Repeat crossmatch on pre & posttransfusion samples with affected unit, and any other units transfused before this reaction • Repeat antibody screening on pre and posttransfusion units

39. Acute Hemolytic Transfusion Reaction • Pathophysiology: • Reaction of preformed Ab (usually ABO) with transfused cells. • Ab coats transfused cells, this stimulates complement system to the membrane attack complex (C5-9) and results in Intravascular Hemolysis. • This leads to a cytokine storm which stimulates the coagulation system resulting in DIC.

40. Clinical Presentation • Occurs within 4 hrs of transfusion • Hypotention, possibly shock, ARF • Sings and Symptoms: (bold = common) • Dark urine (hemoglobinuria) • Persistent hypotention • DIC (oozing) • Fever • Severe costovertebral pain / Pain at infusion site • CP, sense of impending doom • Urticaria, hives, flushing • Vomiting, diarrhea • Hyperbilirubinemia ~ 5-6 hrs post transfusion

41. Management • STOP transfusion • Fluids/Pressors • Lasix (manitol) to continue urinary flow and prevent ATN • Screen and treat DIC • Methylprednisolone 125mg Q6H IV • Or Dex 4mg Q6H IV

42. Delayed Hemolytic Transfusion Rxn • Anamnestic antibody production • Preformed antibody is absent, or present in a very small amount in the recipients pretransfusion blood sample • Recipient has been previously sensitized and therefore has lymphocytes that are primed to respond when re-challenged with the antigen

43. Clinical presentation: • Occur 5 to 14 days after transfusion • Rarely involves complement therefore and hemolysis is extravascular • More subtle presentation: • Unexplained anemia • Failure to achieve expected posttransfusion Hb • Unexplained increase in unconjugated bilirubin with few associated symptoms • Or unrecognized • Management • Supportive care if required • Mainly you need to contact the blood bank so they can identify the alloantibody

44. Febrile (Nonhemolytic) Transfusion Rxn • Pathophysiology: • Ab in recipient reacts against donor WBC, white cell stroma or platelets • Biologic response modifiers – accumulate with storage, cytokines which are leukoctye derived or platelet derived • Plastic can activate complement which in turn activates WBC • Leukocyte reduction has no impact of rxn from plt or Complement

45. Clinical Presentation Management Antipyretics Meperidine Antihistamines really only work as sedative, since mast cells are not involved • Also occurs within four hours of transfusion • Rigors, fever (>38C, or rise > 1C) • Diastolic hypertention • Possibly tachycardia, palpitations, cough • But no hypotention

46. Allergic (Urticarial) Reaction Pathophysiology Management possibly rate dependent, safe to restart at slower rate once rash subsided do not restart if rash is extensive or there is peri-orbital swelling, laryngospasm also do limited TR W/O to R/O hemolysis antihistamines does not affect future transfusions • Hypersensitivity to allergens in transfused unit • 2nd to remnant plasma • can be eliminated by washing cells, or frozen deglycerolized RBC’s • Clinical Presentation: • Urticarial rash, hives • peri-orbital swelling • laryngospasm

47. Anaphylactic Reaction • Pathophysiology: • plasma-containing product transfused to an individual with a pre-existing antibody directed against an epitope in the donor plasma • Ag-Ab complex triggers mast cell degranulation etc • Most often occurs in an IgA deficient recipient when pre-formed anti-IgA is transfused • Anti-IgA is naturally occurring • Donor IgE vs corresponding allergen in patients blood (ex penicillin)

48. Clinical Presentation Management Manage as anaphylactic reaction secondary prevention verify is recipient IgA neg If anti-IgA demonstrated subsequent donations of plasma containing produces from IgA deficient donors, or frozen deglycerolized RBCs or washed (3liters) pRBC • Acute respiratory distress • Laryngeal edema • hypotention

49. Transfusion-Related Acute Lung Injury • Noncardiogenic pulmonary edema associated with passive transfer of donor granulocytes or HLA antibodies • donor antibodies react with neutrophils in recipients lung which results in aggregation and activation in lung microvasculature • Altered vascular permeability resulting in a Pulmonary capillary leak syndrome • Occurs with any plasma containing product • except IVIG, albumin, Rhogam, or coagulation factors (prob related to diluted out by massive donor pool) • Risk is 1:2000 to 1:5000 • Represents 13% of Transfusion related deaths

50. Canadian consensus conference from 2004 on TRALI • Acute onset respiratory distress & hypoxemia within 6hrs after the end of the transfusion with clinical/radiologic evidence of pulmomary edema • Also hypotention, fever • Difficult to differentiate from ARDS • Diagnosis of exclusion • Fluid overload • Cardiac failure • BNP to differentiate TACO from TRALI