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 ( 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
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. (Kell, MNS, Kidd, Duffy, Lutheran)
Pre-transfusion Compatibility Testing (First you need to understand what you are testing for.)
Blood Group Antigen
Carbohydrate
ABO
(H, Lewis, I, P)
Polypeptide Rh
(Kell, MNS, Kidd, Duffy, Lutheran)

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
Forward ABO Typing
Reverse ABO Typing
Rh Typing
(Alloantibody identification if positive)
Alloantibody Detection
Component Selection
The Crossmatch

24. The Forward & Reverse ABO Typing
Forward: Tests recipients red cells.
Reverse: Tests recipients serum to find out what Ab are pesent.
One drop of recipients red cells.
One drop of commercial “anti-A” or “anti-B”.
(facilitate Ab bridging)
Centrifuge.
Gently resuspend and observe for agglutination.
Two drops of recipient serum.
One drop of commercial red cells with A1 or B.
Centrifuge.
Gently resuspend and observe for agglutination

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 Test Checking 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
Immediate Spin Phase
1 drop of each of screening red cells mixed with 2 drops of pts plasma.
Do “immediate spin” reading.
(like ABO)
Tests Abs react at Rm T°.
Ie IgM cold Abs.
37 C Phase
± low-ionic strength saline.
Incubate.
Antiglobulin Phase
Wash tubes ~3 times.
Add antiglobulin reagent.
(Which is a rabbit IgG Ab that recognize the Ig Fc region)
Look for agglutination.

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 Recipient Blood Group Red Cells to Transfuse
Plasma to Transfuse A
A or O
A or AB B
B or O
B or AB AB
AB or O O
Only O
A, B, AB or O

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

46. Allergic (Urticarial) Reaction
Pathophysiology
Management
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
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

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
Acute respiratory distress
Laryngeal edema
hypotention
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

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

51. Management Stop transfusion and provide supportive care
Inform blood bank
Identify donors who are at risk for precipitating TRALI, therefore require prompt notification to the donor center, to identify donor and assess their suitability for future transfusions.
Primary prevention strategies:
Only male donors
Deferral of multiparous women for plasma donation
Testing of multiparous women for Abs
Does not affect future transfusions

52. Septic Transfusion Reaction
Contamination process:
Donor has asymptomatic bacteremia
Skin flora during phlebotomy
Contamination during processing
Accounts for 16% of transfusion associated deaths, ¾ of which are from platelets
high risk 2nd to storage at Rm T
usually gram positive
associated mortality of 25%
RBC
Usually gram negative (endotoxins transfused to patient)
Mortality up to 70%

53. Clinical Manifestations Management
Fever
chills
rigors
shock
Return implicated unit for culture and gram stain
Blood bank needs to notify supplier
Culture patient
Treat patient

54. Transfusion-Transmitted Infectious Disease (In Canada)
HIV – 1 per 7.8 million donations
HCV - 1 per 2.3 million
HBV - 1 per 153,000