A Very Basic Approach to some
of Transfusion Medicine
Christina Lee
R5 Haematology
What are the blood products we use?
The comprehensive list:
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Packed red blood cells (pRBC)
Platelets
Frozen Plasma
Cryoprecipitate
Plasma Derivatives:
– Albumin
– Intravenous immune globulin (IVIG)
– Factor concentrates (Factor VIII & IV)
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.
What is Allo-immunization?
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
Blood Component Preparation
Anticoagulant
used is citrate
based.
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.
Frozen Plasma (in Canada its never “Fresh”)
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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.
Cryoprecipitate
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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.
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.
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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.
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.
So what happens when you
order a unit of blood?
Pre-transfusion Compatibility Testing
(First you need to understand what you are testing for.)
Blood Group
Antigen
Carbohydrate
Polypeptide
ABO
Rh
(H, Lewis, I, P)
(Kell, MNS, Kidd,
Duffy, Lutheran)
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.
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.
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).
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.
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
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
Compatibility Testing – An Intro
1. 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.
2. The antibodies those substances stimulate
and then react with.
I.E. the alloantibodies that are formed against
that antigen (ie not autoantibodies).
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.
Overview of the Type, Screen & Cross Match
Forward ABO
Typing
Reverse ABO
Typing
Alloantibody
Detection
Rh Typing
(Alloantibody
identification
if positive)
Component
Selection
The
Crossmatch
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.
Two drops of recipient
serum.
One drop of commercial
“anti-A” or “anti-B”.
One drop of commercial
red cells with A1 or B.
Centrifuge.
(facilitate Ab bridging)
Gently resuspend and
observe for agglutination.
Centrifuge.
Gently resuspend and
observe for agglutination
Grading Red Cell Agglutination
(The Immediate Spin Technique)
What This Looks like in Real Life
Rh Typing
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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.
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.
Antibody Detection Test – The Process
Immediate Spin
Phase
37 C 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.
• ± 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.
The Antibody Detection Test was
Positive!
Identification of the Antibody
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.
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
AB
O
Only O
A, B, AB or O
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.
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)
Transfusion Reactions
What are the different types?
What is the general management
approach?
STOP the Transfusion
• 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
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.
Clinical Presentation
• Occurs within 4 hrs of transfusion
• Hypotention, possibly shock, ARF
• Sings and Symptoms: (bold = common)
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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
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
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
• 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
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
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
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
• possibly rate dependent,
safe to restart at slower rate
once rash subsided
• do not restart if rash is
extensive or there is periorbital swelling,
laryngospasm
• also do limited TR W/O to
R/O hemolysis
• antihistamines
• does not affect future
transfusions
• Clinical Presentation:
– Urticarial rash, hives
– peri-orbital swelling
– laryngospasm
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 preformed anti-IgA is transfused
– Anti-IgA is naturally occurring
– Donor IgE vs corresponding allergen in patients blood (ex
penicillin)
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
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
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
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
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%
Clinical Manifestations
Management
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• Return implicated unit
for culture and gram
stain
– Blood bank needs to
notify supplier
• Culture patient
• Treat patient
Fever
chills
rigors
shock
Transfusion-Transmitted
Infectious Disease (In Canada)
• HIV – 1 per 7.8 million donations
• HCV - 1 per 2.3 million
• HBV - 1 per 153,000