Second Year Medical Student
Fall Pathology Lab:
Transfusion Medicine and
Blood Banking
October 21st and 22nd 2013
1
Outline, Presenters, Contact Info
• Blood Donation, Component preparation, and Transfusion indications
– Dr. Dan Waxman, M.D.
• Executive Vice President and Chief Medical Officer of Indiana Blood Center
• Clinical Professor of Pathology
• Dwaxman@indianblood.org
• Ordering a blood transfusion
– Dr. Connie Danielson, M.D, PhD
• Chief of Laboratory Service at Wishard Hospital
• Clinical Associate Professor
• Cdaniels@iupui.edu
• Basic principles of transfusion
– Dr. Julie Cruz, M.D.
• Associate Medical Director of Indiana Blood Center
• Volunteer Clinical Assistant Professor of Pathology
• JCruzMD@indianablood.org
2
Outline, Presenters, Contact Info
• Transfusion reactions
– Dr. Nicole Hubbard, M.D.
• Microbiology fellow
• Nbrammer@iupui.edu
• Case Presentation
– Dr. Ted Kieffer, M.D., M.S.
• PGY3 Pathology resident
• TKieffer@iupui.edu
• Presentors
– Dr. Morgan McCoy, M.D.
• PGY3 Clinical Pathology resident
– Dr. Stephanie Slemp, M.D.
• PGY2 Pathology resident
3
Transfusion Medicine Lab
Blood Donation, Component Preparation,
and Transfusion Indications
Transfusion therapy is a set of processes, not just a product
Medical reason to TX
Recruit donor
Screen donor
Pre-TX testing
Collect unit
Issue unit
Prepare components
Administer at bedside
Monitor & evaluate
Infectious diseases
testing
Product: Blood safety
Entire process: Blood transfusion safety
After S. Dzik, MD Blood Transfusion Service, MGH, Boston
Whole Blood
A. Description:
500+/- 50 ml mixed with 70 ml CPD
B. Storage:
21 days stored at 1-6º
C. Indications:
Recently used in military hospitals in combat
areas settings
Proposed clinical trials to examine feasibility
and efficacy in civilian setting
Packed Red Blood Cells
A. Description:
1. 200 ml of RBC with 111 ml of additive solution
2. Packed cell volume = 60%
B. Storage = 42 days
Packed Red Blood Cells
C. Indications:
1. Acute blood loss exceeding 15-20% of blood volume
(pediatric patients - 10-15 ml/Kg) and failure to obtain
hemodynamic stability with reasonable volume of crystalloid
and/or colloid solutions
2. Acute blood loss of any amount if there is clinical
evidence of inadequate oxygen carrying capacity
Packed Red Blood Cells
C. Indications: (Cont’d)
3. Hemoglobin of ≤ 7 gm/dl (hematocrit ≤ 21%), if not due to a
treatable cause (treatment of underlying case is preferable if
patient is not symptomatic)
4. Symptomatic anemia regardless of hemoglobin level
5. Hemoglobin ≤ 8 gm/dl (hematocrit ≤ 24% ) and acute cardiac
disease / or shock
Packed Red Blood Cells
D. Contraindications:
1.
2.
3.
4.
For volume replacement
In place of a hematinic
To enhance wound healing
To improve general “well-being”
Leukocyte-Reduced Red Blood Cells
A. Description:
Packed red cells with leukocytes reduced
(residual leukocyte count less than 5 X 106)
Leukocyte-Reduced Red Blood Cells
B. Processing of Product:
1. Product made during transfusion with filter attached to unit
2. Pre-storage leukocyte reduction at blood center
Leukocyte-Reduced Red Blood Cells
C. Indications:
1. Prevention of HLA/WBC alloimmunization
2. Prevention of recurrent non-hemolytic febrile reactions
3. Prevention of CMV transmission in select groups of patients
Saline Washed Red Blood Cells
A. Description: packed red cells washed with saline
1. 99% of plasma proteins are removed
2. 85% of leukocytes are removed
3. Post-wash K + is 0.2 meq/L
Saline Washed Red Blood Cells
B. Processing: manual and automatic methods
C. Storage: once washed, 24-hour outdate
Saline Washed Red Blood Cells
D. Indications:
1. History of allergic or febrile reactions secondary to plasma
proteins not prevented by pre-transfusion administration of
antihistamines and leukocyte reduction
2. IgA deficiency with documented IgA
antibodies
3. History of anaphylactic reaction to blood components
Irradiated Blood Products
A. Products irradiated:
Whole blood, packed red cells, platelets and
granulocyte concentrates
Irradiated Blood Products
B. Indications: preventing graft
1.
2.
3.
4.
5.
versus host disease
Immunocompromised patients
Directed donations from blood relatives
Premature infants ≤ 1200 gms
Fetuses receiving intrauterine transfusions
Neonatal exchange transfusions
Irradiated Blood Products
C. Processing and final product:
1. Irradiate with 2500cGy
2. Mitotic capacity of lymphocytes is reduced or eliminated
without significant functional damage to other cellular
elements
Irradiated Blood Products
D. Storage:
Red cells outdate 28 days from irradiation (or original
expiration if less than 28 days)
Platelet Concentrates
A. Description:
1. Random donor unit contains 5.5 X 1010 platelets suspended
in 30-50 ml of plasma
2. Apheresis donor unit contains
3. 3.0 X 1011 platelets suspended in 300-400 ml of plasma
2-RBCs
Plateletpheresis
Plasmapheresis
Platelet Concentrates
B. Storage:
1. Stored at 20-24º C on a rotator
2. 5-day outdate
Platelet Concentrates
C. Indications: prevention and cessation of bleeding
1. Severely thrombocytopenic (less than 10,000 or 20,000
depending on institution)
2. Moderately thrombocytopenic and bleeding (less than
50,000)
3. Surgery or invasive procedure (less than 50,000)
4. Diffuse microvascular bleeding following cardiopulmonary
bypass or with intra-aortic balloon pump (less than 100,000)
5. Bleeding with qualitative platelet defect
6. Massive Transfusion Protocols (MTP)
Platelet Concentrates
D. Contraindications:
1. Idiopathic Thrombotic Thrombocytopenic Purpura (ITP)
2. Thrombotic Thrombocytopenic Purpura (TTP)
Platelet Concentrates
E. Dosage:
1.
2.
3.
4.
4-6 platelet concentrates
1 apheresis unit
Platelet count should increase 25,000 – 30,000/cc3
Each dose has equivalent of one unit of fresh plasma*
* Unless using platelet additive solution (PAS)
Frozen Plasma
A. Description:
1. 225-275 ml of plasma and CPDA-1, including 25 meq of citric
acid
2. Jumbo plasma 400 cc or greater
3. Frozen within 8 hrs = FFP
4. Frozen within 24 hrs = 24FP
B. Storage: 1 year at -18º C
Frozen Plasma
C. Outdate once thawed (1-6º C)
1. 24 hours for FFP or 24FP
2. 72-120 hours if relabeled as Thawed Plasma
37
Frozen Plasma
D. Indications:
1. Treatment of coagulopathy due to clotting factor deficiencies
2. Patient is bleeding actively with PT and/or PTT greater than
1.5 normal (INR > 1.8) and platelet count above 50,000
3. Coumadin overdose with major bleeding or impending
surgery
4. Treatment of TTP
5. Massive Transfusion Protocol (MTP)
Frozen Plasma
E. Contraindications:
1. Volume expansion
2. Treatment of nutritional deficiencies
Cryoprecipitate
A. Description: each unit consists of 10-30 ml residual plasma
than contains 80 units of factor VIII: C and 250 mg of
fibrinogen
B. Storage: 1 year at -18º C
Cryoprecipitate
C. Indications:
1.
2.
3.
4.
Hypofibrinogenemia (≤ 100 mg/dl)
Dysfibrinogenemia
Factor XIII deficiency - rare
MTP
New/Future Products
A. Growth Factors to stimulate cell production
B. Sterilized products to prevent infections*
C. Synthetic products to replace current transfusions*
*Approval delayed due to regulatory reluctance.
Questions
?
TRANSFUSION MEDICINE LAB
• ORDERING A BLOOD TRANSFUSION:
• What does your patient need?
– Why? How much? When? Alternatives?
• Obtain Consent
• Write Order
• Obtain Specimen and send to Blood Bank
Case Presentation
The patient is a 64 year old man 2 days after a total
hip arthroplasty. He has a history of coronary artery
disease and had a MI 5 years ago. He complains that
during physical therapy he becomes short of breath
and develops chest pain which resolves with rest and
sublingual nitroglycerine.
Today his Hgb/HCT= 7.5 gms/dL/23%.
You decide to order 1 unit of packed red blood cells
to be transfused. Why?
packed Red Blood Cells
• Indication – patient is anemia AND symptomatic
• Hgb < 8gm/dL
• Patient complaining of shortness of breath
*1 unit RBC/Adult increases Hgb 1 gm/dL*
• Since the 1990’s it has become clear that
“over-transfusion” can increase morbidity and
mortality.
Transfusion Guidelines:
• In the USA currently accepted guidelines for
transfusion of red cells is generally:
– Hgb ≤7 g/dL many need transfusion
– Hgb 7-9 g/dL variable need for transfusion
– Hgb≥ 10 g/dL very few need transfusion
Blood is a drug with a variable safety profile
(discussed later)
Normal levels of Hgb in adults
• Men: 13.8 to 18.0 g/dL
• Women: 12.1 to 15.1 g/dL
• If the concentration is below normal, this is called anemia.
• Hematocrit, the proportion of blood volume occupied by red blood
cells, is typically about three times the hemoglobin concentration
measured in g/dL.
Blood volume of an Adult vs a Neonate
Equations
Hematocrit (HCT)
Ex: 40%
An adult has
=
=
~ 2L of RBC
~ 3L of plasma
RBC volume
Total blood volume
? RBC
2 liters
volume
5 liters
Obtain Consent:
•
•
•
•
Written informed consent
Expected benefits
Possible risks
Alternatives
• If transfusion is refused -document
Consent form
56
Obtain written informed consent:
What are the patient’s concerns?
What are the Blood Bank’s concerns?
Complications of Transfusion
Complications of Transfusion continued
Write the order:
Type and cross patient for 1 unit of red blood cells.
Give when ready over 2-4 hours as tolerated
Obtain Specimen:
✓Identifiers on wrist band
✓Identifiers on specimen label
✓Identifiers on requisition
All Identical
A Patient’s Specimen must have:
•
•
•
•
Patient identifiers
Phlebotomist signature
+/- witness signature
Date and time collected*
*A patient’s specimen can be used for
3 days
Questions:
A unit of red blood cells should raise the
Hgb concentration in a nonbleeding adult
by approximately:
1) 0.5 grams/dL
2) 1.0 grams/dL
3) 2.0 grams/dL
4) 5.0 grams/dL
Questions:
A unit of red blood cells should raise the
Hgb concentration in a nonbleeding adult
by approximately:
1) 0.5 grams/dL
2) 1.0 grams/dL
3) 2.0 grams/dL
4) 5.0 grams/dL
Questions:
A patient’s specimen sent to Blood Bank
may be used for testing for :
1) 12 hours
2) 24 hours
3) 2 days
4) 3 days
Questions:
A patient’s specimen sent to Blood Bank
may be used for testing for :
1) 12 hours
2) 24 hours
3) 2 days
4) 3 days
Questions:
A patient with a Hgb concentration of 10
g/dL would have a HCT of approximately:
1) 20%
2) 20 g/dL
3) 30%
4) 31 g/dL
Questions:
A patient with a Hgb concentration of 10
g/dL would have a HCT of approximately:
1) 20%
2) 20 g/dL
3) 30%
4) 31 g/dL
Questions
?
Basic Principles of
Transfusion
ABO, Rh, Other Blood Groups
Pretransfusion Testing
ABO Blood Groups
• 4 Types: A, B, AB and O
the name of the blood group corresponds to the presence
of the antigen associated with the sugar residue (ie, a
person whose red blood cells carry the A antigen has
blood group type A. Type O red blood cells lack these
terminal sugars, and the corresponding antigen is called
the “H” antigen.
Antigen Specificity
Immunodominant sugar
H
L-fucose
A
N-acetylgalactosamine
B
D-galactose
ABO Antigens and Antibodies
Blood Group
Antigen on Red
Blood Cell
Antibodies in serum
Group O
H (neither A nor B) Anti-A, Anti-B
Group A
A
Anti-B
Group B
B
Anti-A
Group AB
A and B
Neither anti-A nor
anti-B
• ABO compatibility is the primary concern when selecting a unit of blood
for transfusion
• Other antigens are present on red blood cells, and antibodies to these
antigens are also important. However, these antibodies occur only after
the individual has been exposed (or sensitized) to the antigen through
transfusion or pregnancy. This is also called alloimmunization.
RED CELLS
RECIPIENT CELLS
TRANSFUSED (DONOR) CELLS
PLASMA
RECIPIENT CELLS
TRANSFUSED (DONOR) CELLS
ABO Frequencies
Group O
45%
Group A
40%
Group B
11%
Group AB
4%
ABO Typing
• forward type involves testing the patient’s red blood cells with
commercial anti-sera containing anti-A and anti-sera containing anti-B.
Reactivity with the specific anti-sera indicates that antigen is present on
the patient’s red blood cells
• reverse type combines the patient’s serum with reagent red blood cells
that are either type A or type B. Reactivity indicates the patient has
circulating antibody to the corresponding antigen on the reagent red
blood cell.
• Reactivity occurs in the form of agglutination – which is the clumping
together of the red blood cells resulting from the interaction of the
antibody and its corresponding antigen. This clumping can be observed
visually or detected photometrically in automated equipment. Strength
of agglutination is graded from 0 (no agglutination) to 4+ (very strong).
ABO Typing sera and reagent red cells
Agglutination
4+Agglutination (strong positive)
0 agglutination (negative)
ABO Forward and Reverse Groupings
Forward group: Patient’s cells with
reagents
Reverse Group: Patient’s serum with
reagents
Blood
Group
Anti-A
Anti-B
Antigen(s)
on RBCs
A1 cells
B cells
Antibody(ies) in
serum
O
0
0
No A or B
+
+
A and B
A
+
0
A
0
+
B
B
0
+
B
+
0
A
AB
+
+
A and B
0
0
No A or B
The Rh Blood Group System
• RhD
• The RhD gene is either present or absent – it’s absence is denoted by
the placeholder “d”. There is no “d” gene, and no “d” antigen.
• An individual who possesses no copies of the RhD gene (did not inherit
RhD from either parent) is indicated by d, and said to be “Rh negative”.
Eighty-five percent of Caucasians express the D antigen.
• Some RBC’s exhibit a weaker than normal form of the D antigen, giving
weaker or negative reactivity with standard anti-D reagent. This antigen
expression is known as weak D.
RhCE
• The RhCE gene codes for either RhCe, RhcE, Rhce, or RhCE proteins.
Again, these genes are inherited as a haplotype from each parent. The
following table shows the possiblehaplotypes and their frequencies
utilizing Fisher-Race and Weiner terminology.
Prevalence (%)
Haplotype
Fisher-Race
DCe
dce
DcE
Dce
dCe
dcE
DCE
dCE
Haplotype
Weiner
R1
r
R2
R0
r’
r”
Rz
ry
White
Black
Asian
42
37
14
4
2
1
<0.01
<0.01
17
26
11
44
2
<0.01
<0.01
<0.01
70
3
21
3
2
<0.01
1
<0.01
Calculating prevalence
• Since one haplotype is inherited from each parent, the
phenotype frequencies may be obtained by multiplying the
haplotype frequencies for the population. For example, the
frequency of the dce/dce (rr) phenotype in the Caucasian
population is .37 X .37 = .14 or 14%. So, if an alloimmunized
individual required red blood cells of this type for
transfusion, approximately 14% of randomly collected
donor units would be appropriate.
• What frequency would you expect to find the dce/dce (rr)
phenotype in the Black population?
Other Blood Group Systems
• Lewis
– Antibodies to the antigens in the Lewis system are rarely clinically
significant. The antigens are not synthesized on the red cells, but are
absorbed from the plasma. The importance of the Lewis system is more its
association with the Se gene and the determination of an individual’s
secretor status.
• Kell
– After ABO and Rh, the K antigen is the most immunogenic blood group
antigen causing both HDFN and delayed hemolytic transfusion reactions.
About 10% of whites and 2% of blacks are positive for the K antigen. The
antithetical antigen, k, (pronounced Cellano) is a high incidence antigen,
meaning <1% of the population lacks this antigen.
Other Blood Group Systems
• Kidd
– Two antigens, Jka and Jkb are responsible for the common phenotypes. The
Kidd antigens are located on a membrane glycoprotein involved in urea
transport in the red cell. They are often weakly reacting antibodies showing
dosage. Dosage refers to stronger expression of the antigen when the
individual inherits the gene homozygously, vs. heterozygously. It is
important that antibodies to these antigens are ruled out on homozygous
reagent cells, since a heterozygous cell may be nonreactive due to the low
expression of the antigen. Kidd antibodies are notorious for weakening over
time and even becoming undetectable. Despite this, they cause severe
delayed hemolytic transfusion reactions. They are also implicated in HDN,
but it is usually a milder form.
Other Blood Group Systems
• MNS
– The most important antigens that affect transfusion are M, N, S, s and U.
The M and N antigens are carried on glycophorin A, and S,s, and U are
carried on glycophorin B. Anti-M and anti-N can occur as naturally occurring
agglutinins (IgM) and as such usually react at room temperature or below.
They do not cause HDFN. These antibodies can usually be ignored for
transfusion, unless they are also reactive at 37 C due to the presence of an
IgG component. In contrast, S and s are IgG reactive at 37 C, are clinically
significant and cause HDFN. Anti-U is a rare antibody which is associated
with HDFN and hemolytic transfusion reactions, usually seen in the African
American population. It occurs in individuals who are S-s-U-, and individuals
who have anti-U are incompatible with greater than 99% of the donor
population.
Other Blood Group Systems
• Duffy
– The Duffy system is made up of six antigens that reside on an acidic
glycoprotein . The glycoprotein carrier molecule is also known as the Duffy
antigen receptor for chemokines (DARC) and functions as a cytokine
receptor. It is also a receptor for the malarial parasite Plasmodium vivax.
The null phenotype Fy(a-b-) is rare in Caucasians but common in blacks. It is
thought that this developed as a survival advantage in malarial regions of
Africa, since such individuals are naturally resistant to infection by P. vivax.
Anti-Fya is a relatively common antibody, while anti-Fyb is less common.
Both are clinically significant and associated with hemolytic transfusion
reactions and HDFN.
Antibody Screen
The goal of antibody screening is to detect unexpected clinically significant
red cell antibodies. In general, clinically significant antibodies are
antibodies known to have caused hemolytic transfusion reactions,
hemolytic disease of the fetus and newborn (HDFN), or shortened survival
of transfused red blood cells. The method used must be capable of
detecting clinically significant antibodies, which requires that the antibody
screen method include a 37 C incubation with reagent red cells followed by
an indirect antiglobulin test (IAT).
The Indirect Antiglobulin Test (IAT):
The IAT is used to detect in-vitro sensitization and detects anti-red
cell antibodies in patient’s serum or plasma. Procedural steps are as
follows:
1. Patient’s plasma or serum is incubated at 37 C with red cells
(screen or panel cells of known antigenic composition, ie.
reagent red cells, or donor cells as in the crossmatch).
2. A potentiator (LISS, PEG) may or may not be added
3. During incubation, if an antibody is present in the plasma or
serum and the corresponding antigen is present on the red cells,
the cells become sensitized by the antibody adsorbing to
antigens on the red cell surface
4. After incubation, the red cells are washed with saline to remove
unbound antibody
5. Antihuman globulin serum (anti-IgG or polyspecifc AHG, usually
the former) is added and forms RBC agglutinates if the antibody
has attached to the antigen sites during incubation
Indirect antiglobulin test
Crossmatch Methods:
• Immediate spin crossmatch:
The immediate spin (IS) crossmatch is performed only after an antibody
screen is done and found to be negative on a current specimen. It is
performed at room temperature without enhancement. The patient
should have no history of clinically significant antibodies. The
immediate spin crossmatch is meant to detect ABO incompatibility. It
can also detect cold reactive (generally clinically insignificant)
antibodies that react at room temperature.
• Antihuman Globulin (AHG) crossmatch
The AHG is performed as described for the IAT above, and tests the
patient’s serum or plasma against the donor RBCs intended for
transfusion to the patient. It will detect ABO incompatibility as well as
other clinically significant antibodies, such as antibodies in the Rh,
Kell, Kidd, and Duffy systems.
Computer-Assisted (Electronic) Crossmatch
Principle:
• Using a validated computer system, patients with no history of clinically
significant antibodies and a negative antibody screen on the current
specimen are issued ABO specific or compatible donor units. Specific
conditions must be met:
–
–
–
–
–
Two independent determinations of the ABO group of the patient
No history of clinically significant antibodies
A negative antibody screen on a current specimen
Donor units are ABO confirmed
The computer system is validated on site, results are entered directly into
the computer, and there is logic in the system to recognize incompatibility
In the last section:
• You ordered type and cross for 1 unit of red blood cells for your patient.
• Now, you are the blood bank technician:
• You receive an appropriately labeled and collected specimen with an
order to type and cross for one unit of red blood cells. First, you
perform the ABO and Rh Typing:
Forward type: tube on left contains anti-B, tube on right
contains anti-A.
Reverse type: Tube on left contains A1 Cells, Tube on right
contains B cells
Forward
Anti-A
Reverse
Anti-B
Type?
A Cell
B Cell
Type?
Patient RBC with anti-D
Rh Typing
Anti-D
• What is the patient’s ABO/RH?
Antibody screen
• A 3 cell antibody screen is performed and
two of the three cells are positive. What
does this mean?
You test the patient’s serum against a panel of reagent red
blood cells with the following results:
• Antibody is present to what antigen?
• What does this imply for transfusion?
• A unit of RBCs that is Type A, Rh positive
and confirmed negative for the Jkb antigen
is selected for the patient. The AHG
crossmatch is performed and the unit is
compatible. Is this unit appropriate for the
patient?
Questions
?
Transfusion Reactions
Nicole Hubbard M.D.
Microbiology fellow
Outline
• Definition
• Types of transfusion reactions
– Acute febrile transfusion reactions
– Acute non-febrile transfusion reactions
– Delayed febrile transfusion reactions
– Delayed non-febrile transfusion reactions
• Risk of Transfusion reactions
• Approach and follow-up to suspected transfusion reactions
105
Definition
• Unfavorable events that occur during or after transfusion
• Can happen with any blood component
• PRBC, Plasma, Platelets, Cryoprecipitate
• Rho (D) immune globulin, IVIG, Factor concentrate
• Stem cells, Human albumin, Granulocytes
Categorization of transfusion reactions
Febrile
Afebrile
Acute
Hemolytic (HTR)
TRALI
Bacterial
contamination
Non-hemolytic
(FNHTR)
TACO
Urticarial/allergic
Premedicated febrile
Delayed
Hemolytic (DHTR)
TA-GvHD
PTP
Iron overload
Acute hemolytic transfusion reactions
(AHTR)
• Most severe reaction (may be fatal!)
• All reactions are hemolytic until proven otherwise
• ABO incompatibility most commonly from clerical
error
• IgM antibodies bind to the RBCs → complement
activation → formation of MAC → RBC lysis
• Coagulation cascade activation from Ag-Ab
complexes →
• Intravascular thrombi → schistocytes
• Coagulation cascade and complement → cytokine
production → additional symptoms
• Treatment is supportive
Signs and Symptoms
• Fever/Chills 1°C or 2° F
(most common)
• Tachycardia
• Hypotension
• Bleeding
• Dyspnea
• Impending doom
• Nausea/Vomiting
• Pain: flank, back, chest,
head, infusion site
• Dark urine
(hemoglobinuria)
• Death
Laboratory Findings
• Hemoglobinemia (pink or red serum/plasma)
• Hemoglobinuria (NOT hematuria)
• Usually positive direct antiglobulin test (DAT) but can be
negative (all Ab coated cells already lysed)
• Elevated indirect bilirubin and LDH
• Decreased haptoglobin
• Hyperkalemia
• Peripheral smear: Schistocytes
Transfusion Related Acute Lung Injury
(TRALI)
• #1 cause of fatal transfusion reactions in the US
• Usually donor anti-HLA or anti-neutrophil antibodies attach
to neutrophils in lung → neutrophil activation → vascular
damage → edema and impaired gas exchange
• Begins during or within 6 hours of transfusion
• No pre-existing acute lung injury
• Prevented by use of male plasma
– Why???
Symptoms and Other Findings
•
•
•
•
•
Dyspnea
Fever
Tachycardia
Hypotension
Frothy endotracheal
aspirate
• Death
• CXR shows whiteout/edema
Suspected TRALI
• Donor testing for anti-HLA and anti-neutrophil antibodies
• Permanent deferral for donor if positive
Bacterial Contamination
• Contaminated blood product → sepsis
• Most common in platelet products
– Why???
• Symptoms:
– Rapid high fever, hypotension, tachycardia, and shock
– May also have violent rigors, nausea, vomiting, diarrhea
– Shock and death
Febrile non-hemolytic transfusion
reactions (FNHTR)
• Most frequently reported
• Temperature increase of >1°C or >2°F with no other
explanation
• Cytokines from WBC in product during storage or antibody
activation of WBC
• Usually mild symptoms
– Fever
– Chills (may be only symptom if pt is premedicated)
• Treatment:
– Antipyretics (acetaminophen)
– Leukoreduced products (pre-storage)
Categorization of transfusion reactions
Febrile
Afebrile
Acute
Hemolytic (HTR)
TRALI
Bacterial
contamination
Non-hemolytic
(FNHTR)
TACO
Urticarial/allergic
Premedicated febrile
Delayed
Hemolytic (DHTR)
TA-GvHD
PTP
Iron overload
Transfusion associated circulatory
overload (TACO)
• Fluid overload precipitated by blood product
• Risk factors: Renal impairment and heart failure
• Signs and symptoms: Dyspnea, tachycardia, hypertension,
jugular venous distension
• Treatment: Support respiratory function and diuretics
TRALI v. TACO
TRALI
• Dyspnea
• Tachypnea
• Tachycardia
• Hypotension
• Fever
TACO
• Dyspnea
• Tacypnea
• Tachycardia
• Acute Hypertension
• Jugular venous distension
• Rales
• BNP levels
Allergic Reaction
• Second most common type of transfusion reaction
• Only reaction that allows the restart of procedure if
symptoms resolve
• Caused by type I hypersensitivity to donor plasma proteins
• Symptoms: Pruritis, urticaria, erythema
• Treatment/Prevention
– Benadryl
– Could give plasma deficient products (washing)
Anaphylactic Reaction
• Most severe allergic reaction
• Symptoms: Rapid onset of laryngeal edema leading to
dyspnea, tachypnea, hypotension, tachycardia, afebrile
• Often associated with recipients that lack IgA AND have
anti-IgA
• Can give washed or IgA deficient products
Categorization of transfusion reactions
Febrile
Afebrile
Acute
Hemolytic (HTR)
TRALI
Bacterial
contamination
Non-hemolytic
(FNHTR)
TACO
Urticarial/allergic
Premedicated febrile
Delayed
Hemolytic (DHTR)
TA-GvHD
PTP
Iron overload
Delayed Hemolytic Transfusion
Reactions (DHTR)
• Anamnestic response =
resurgence of antibody faster
and more severe after second
exposure
• IgG antibodies bind to the
RBCs(sensitized cells) →
processed by reticuloendothelial
system→ RBCs removed by
spleen
• Incompletely removed cells
result in spherocytes
• Main causes Kidd (Jk) and Duffy
(Fy) systems
Signs and Symptoms
•
•
•
•
•
Fever/chills (mild)
Mild jaundice
Anemia/pallor
Splenomegaly
Rarely death
Laboratory Findings
•
•
•
•
•
DAT positive
Hyperbilirubinemia
Usually no free hemoglobin (versus AHTR)
“New” antibody
Spherocytes on peripheral smear
Transfusion-Associated Graft versus
Host Disease
• Highly (> 90%) fatal
• Donor WBC recognize pt’s HLA antigens → WBC activation
→ destruction of host tissues
• Signs and symptoms: Fever, diarrhea, skin rash, bone
marrow suppression
• Treatment/Prevention
– Supportive care
– Irradiation of blood products
• Why???
Categorization of transfusion reactions
Febrile
Afebrile
Acute
Hemolytic (HTR)
TRALI
Bacterial
contamination
Non-hemolytic
(FNHTR)
TACO
Urticarial/allergic
Premedicated febrile
Delayed
Hemolytic (DHTR)
TA-GvHD
PTP
Iron overload
Post-transfusion Purpura
• Patient abs against donor platelet antigens (usually PL-A1)
• PL-A1 negative patients (RARE)
• Signs/symptoms: Profound thrombocytopenia, purpura,
bleeding
• Patient’s own platelets undergo destruction as well
(unknown mechanism)
• Treatment: supportive/plasmapheresis
Iron Overload
• Transfusion dependent anemia (i.e. sickle cell and
thalassemia patients)
• Iron accumulates with multiple transfusions
• Results in end-organ damage
• Treatment/prevention
• Chelation therapy
• Exchange transfusions
Risks of Blood Product Transfusion
•Hives: 1 in 30 to 100
•Febrile reactions: unknown quantity, but common
•Transfusion Associated Circulatory Overload (TACO): 1 in 3,000
to 12,000
•Transfusion-Related Acute Lung Injury (TRALI): 1 in 5,000
•Acute Hemolytic Reactions: 1 in 15,600 to 35,700
•Bacterial Infection: 1 in 20,000
•Hepatitis B: 1 in 200,000
•HTLV-1: 1 in 641,000
•Hepatitis C: 1 in 1.2 million
•HIV: 1 in 1.4 million
•Other infectious diseases such as West Nile virus: less than 1 in
million
•Other infections such as Babesiosis or Chagas: very rare
•Anaphylaxis: 1 in 20,000
Indianapolis Coalition for Patient Safety, Transfusion consent form.
What To Do For Suspected Transfusion
Reaction?
•
•
•
•
•
•
•
Stop Transfusion
Keep line open with saline
Report to physician
Order transfusion work up
Clerical check at bedside
Unit and tubing need to be returned to blood bank
Patient blood sample sent to lab
Laboratory Work-Up
•
•
•
•
Examine blood for hemolysis
Perform DAT
Repeat type and cross
If greater then >2°C, send retention segment and bag for
culture
• Also, blood cultures on patient
• If suspect HTR
• Collect first voided urine
• Check bilirubin 6 hours post transfusion
• Check haptoglobin
• Check CBC
References
• References available upon request.
• Thank you:
• Judi Seidel, MT (ASCP) SBB
• Dr. Steven Gregurek M.D.
Questions
?
Case Presentation
MS2 Pathology Lab
Blood Bank Lab 10/21 & 10/22
Ted Kieffer
PGY3
Case Presentation
• A 25 year old pregnant Chinese female is
brought to the emergency department
following a motor vehicle accident. She does
not speak English but appears to be in
significant pain and grasping her lower
abdomen.
What do you do?
Case Presentation
• History and review of systems cannot be
obtained due to language barrier and no
translator readily available.
• Vitals
–
–
–
–
HR 110 beats per minute
Resp 20 breaths per minute
BP 135/90
Sats 97% on room air
Case Presentation
• Physical exam
– Relatively unremarkable aside from
• Abdominal exam which reveals a band-like contusion
across the lower abdomen and a firm, tender uterus
with a fundal height of 26 cm
• Vaginal exam remarkable for blood
What else might you want to know?
(i.e. who else might you want to evaluate?)
Case Presentation
• Fetal heart rate by doppler – 160 bpm
Reasurring?
Current Differential Diagnosis?
Placental abruption
Preterm Labor
Placenta previa
Uterine rupture
Subchorionic hematoma
Case Presentation
Current Differential Diagnosis:
Placental abruption – acute onset, tender & firm
abdomen/uterus, maternal hypotension
Preterm Labor – gradual onset, progressive, Mucus plug may
be mistaken for vaginal bleeding
Placenta previa – characteristically painless vaginal bleeding at
>20 weeks gestation
Uterine rupture – sudden onset FHR abnormalities, vaginal
bleeding, abdominal pain, and maternal hypotension
Subchorionic hematoma – light vaginal bleeding, generally no
abdominal pain, <20 weeks gestation
Case Presentation
• Lab tests?
–
–
–
–
CBC
BMP + Ca, Mg, Phos
Type and cross
Coag studies
• Including: Fibrinogen, fibrinogen degradation products (FDP), D-dimer
– *other tests
• Alpha fetal protein (AFP), human chorionic gonadotropin (hCG)
• **Kleihauer-Betke test**
• Imaging?
– Ultrasound
• Anything else? Orders?
– Fetal heart rate monitor
– Pain medication for mom
Case Presentation
Abdominal US/FAST scan
Diagnosis!!!
Placental Abruption
Radiologist eventually reads sonogram
and interprets findings as small abruption
Case Presentation
• What do you do now?
– Vitals still stable
– Lab results
• CBC – 10.2
8.9>------<347
31
• BMP – 142/105/10
Ca – 4.9 mg/dL (wnl)
---------------<95 Mg – 2.0 mg/dL (wnl)
4.0/24/1.1
Phos – 3.5 mg/dL (wnl)
Anything else?
Treat Patient – Tocolysis
Magnesium Sulfate
Case Presentation
• What do you do now?
– Lab results
• Coag tests
– PT – 9 seconds (normal 11-13.5 sec)
– aPTT – 21seconds (normal 25-35 sec)
• Fibrinogen – 350 mg/dL (wnl)
• FDP – 5 mcg/mL (wnl)
• D-dimer - <5μg/mL FEU (wnl)
• AFP & hCG wnl
• Kleinhauer–Betke test – 0.2%
Hypercoag?
Why did you order these?
Case Presentation
B-cells
Anti-D
Anti-A
A-cells
Anti-B
• What do you do
now?
– More lab results
• Type and cross
–
–
–
–
ABO type A
Rh negative
Screen positive
DAT positive with
mixed field
DAT
Antibody screen
4+
4+
Case Presentation
Antibody Panel
0
4+
4+
4+
0
0
0
0
0
0
0
M
Anti-D with a mixed field on patient sample
Case Presentation
• At that moment the translator arrives and
informs you the patient is feeling much
better, abdominal pain has decreased, and
that her contractions appear to have ceased.
• Any other questions you would like to ask?
Prior birth history?
Case Presentation
• The translator informs you that the patient
has had two children prior to this pregnancy,
all with the same father. The first was born
without complications but the second was
jaundiced for several weeks. All prenatal and
postnatal care was received in rural China.
Case Presentation
• So what is happening? What must you be
concerned about? What are your next steps?
• Lets bring it all together
– Diagnosis of placental abruption
– Blood tests indicate two different RBC in
mom’s blood
– Mother is Rh negative
– Anti-D antibody identified on RBC panel
– History of complicated pregnancy
FMH
HDFN
Case Presentation
Causes of Fetal Maternal
Hemorrhage (FMH)
Delivery
Indications for administering RhIG if
mother is Rh- and fetus Rh+/unk
At 28 weeks of gestation
Induced abortion
Spontaneous abortion
Spontaneous abortion, threatened abortion, induced
abortion
Ectopic pregnancy
Ectopic pregnancy
Partial molar pregnancy
Chorionic villus sampling
Invasive procedures: genetic amniocentesis; chorionic villus
sampling; multi-fetal reduction; fetal blood sampling
Cordocentesis
Percutaneous fetal procedures (eg, fetoscopy)
Amniocentesis
External cephalic version
Abruptio placenta
Hydatidiform mole
Fetal death in the second or third trimester
Blunt trauma to the abdomen
Antenatal hemorrhage
Maternal abdominal trauma
Antepartum hemorrhage in the second or third trimester
(eg, placenta previa or abruption)
Spontaneous
Manual removal of the placenta
External cephalic version
Case Presentation
• Management of FMH – RhIG
– Kleihauer-Betke test
• standard method of quantitating
fetal-maternal hemorrhage for
calculating RhIG dose
• Maternal blood smear exposed to acid
pH which dissolves adult hemoglobin
and leaves fetal hemoglobin intact –
special stain applied (Shepard’s)
which stains hemoglobin and
percentage of fetal cells is calculated
out of 2000
Case Presentation
• Management of FMH – RhIG
– Using Kleihauer-Betke test
• Calculate maternal blood volume:
– prepregnant wt kg x 70ml/kg x [1.0 + (0.5 x wks gestation/36)] - est blood
loss
– Or estimate 5L
• Calculate fetal blood volume in maternal circulation:
– (% from KBT) x maternal blood volume
• Calculate dose of RhIG
– 1 vial (300μg RhIG)/30mL fetal blood
– fudge factor – round up one vial for values of n.0 to n.4; round up two vials for values
of n.5 to n.9
Case Presentation
• Management of FMH – RhIG
– Using Kleihauer-Betke test in our patient
• Calculate maternal blood volume:
– No info given – using 5L or 5000mL
• Calculate fetal blood volume in maternal circulation:
– (0.2%) x 5000mL = 0.002 x 5000mL = 10mL
• Calculate dose of RhIG
– 1 vial/30mL fetal blood x 10mL = 0.33 vials = 1 vial (rounded up)
– You administer 1 vial of RhIG to the patient
– Why did you go through all of that effort?
Hemolytic Disease of the Fetus/Newborn (HDFN)
Defined as the destruction of fetal and neonatal red blood cells by maternal
antibodies (termed alloantibodies) – Historically IgG Rh/anti-D antibodies
Pathophysiology of HDN
• Maternal IgG antibodies cross the placenta and attach to fetal red blood
cells
• Red blood cells hemolyzed or removed via reticulo-endothelial system
• Resultant anemia causes accelerated production of RBCs by bone
marrow, termed erythroblastosis fetalis
• In severe disease, bone morrow inevitably fall short of necessary RBC
production
• Body responds with extramedullary hematopoesis in the spleen and
liver
• Hepato-splenomegaly causes portal hypertension and hepatocellular
damage
• Anemia coupled with hypoproteinemia leads to massive, diffuse edema
and high cardiac output heart failure
Erythroblastosis and Hydrops Fetalis
Sequelae of HDN
• Hyperbilirubinemia
– RBC destruction does not cease with delivery
• IgG due to its small size as a monomer distributes throughout tissues (intravascular and
extravascular)
• IgG has a half life of 25 days
– Prior to delivery bilirubin is transported across placenta, conjugated by maternal
liver
– Bilirubin conjugation system in a neonate is immature
– Without therapy unconjugated/indirect bilirubin can reach toxic levels (18-20
mg/dL) and diffuse into the brain causing kernicterus and acute bilirubin
encephalopathy
Kernicterus and Acute Bilirubin Encephalopathy
Development of alloantibodies
•
Mother must be antigen negative
–
•
Mother must be exposed to antigen
–
–
–
–
•
Feto-maternal hemorrhage
Transfusion with ABO compatible, minor group incompatible blood
Injection with needles contaminated with minor group antigen positive blood
Minor blood group mismatch allogeneic stem cell transplant
Antigen exposure induces antibody formation
–
–
•
Antigen positive individuals will not form antibodies
Exact volume unknown (varies between individuals) but as little as 0.1 mL of Rh+ RBC have
been shown to stimulate antibody production
Larger volume of exposure tends to produce a more robust response
Following antibody production mother must become pregnant with an antigen
positive fetus
–
The initial response will be IgM which will not cross the placenta
• In most cases, the first minor group incompatibility between mother and fetus will not be
affected, with exceptions
–
Subsequent exposure will induce memory cells to produce IgG antibodies which will in turn
cross the placenta and cause hemolysis or clearance
Development of fetal alloantibodies
• Mendelian genetics
– Genes code for an enzyme (ABO
system), surface protein (Rh
system), or nothing
(silent/amorph)
– Blood group genes major
(ABO)and minor (all others) are
inherited in a Co-dominant
pattern
• Each parent contributes half of
the inheritance
• Individual traits are inherited
independent of each other
Development of fetal alloantibodies
Rh+
Rh
Rh-
Dd
dd
Dd
Rh +
Dd
Rh +
dd
Rh -
dd
Rh -
Development of fetal alloantibodies
Rh +
Rh
Rh-
Dd
dd
Unlike A and B antibodies,
many minor antibodies are
not naturally occurring
First Rh + child sensitizes Mother
Mother develops Anti-D antibodies
Dd
Dd
First
Child
IgG Alloantibodies
cross placenta
Rh +
Second
Child
Second child conceived
Rh +
dd
Rh -
dd
Rh -
Why only IgG antibodies?
• Only IgG type antibodies can
cross the placenta
• Actively transported via a receptor
specific to IgG Fc region
• Starts in the second trimester and
continues until birth
Antigens associated with HDFN
• Major incompatibility
• ABO antigens – usually mild HDFN, A and B antigens less
well developed in neonate (actually protects from minor
incompatibility)
• Minor incompatibility
– Frequently associated with severe HDFN
• Rh (D, c) and Kell (K)
– Infrequently associated with severe HDFN
• See next slide
Infrequently associated with severe disease
Colton
Coa
MNS
Mta
Rhesus
HOFM
Co3
MUT
LOCR
ELO
Mur
Riv
Dia
Mv
Rh29
Dib
s
Rh32
Wra
sD
Rh42
Wrb
S
Rh46
Duffy
Fya
U
STEM
Kell
Jsa
Vw
Tar
Diego
Jsb
Antigens infrequently
associated with severe HDFN
Rhesus
Bea
Other
antigens
HJK
k (K2)
C
JFV
Kpa
Ce
JONES
Kpb
Cw
Kg
K11
Cx
MAM
K22
ce
REIT
Ku
Dw
Rd
Ula
E
Kidd
Jka
Ew
MNS
Ena
Evans
Far
e
Hil
G
Hut
Goa
M
Hr
Mia
Hr0
Mit
JAL
Rh Alloimmunization: diagnosis
• Rh(D) typing and screen
– Performed at first prenatal visit
– If Rh(D)-, screen negative, and prenatal course
uncomplicated
• Repeat at 28 weeks
• Repeat at delivery
– If Rh(D)-, screen positive → diagnostic tests
• Indirect antiglobulin test on maternal serum with titer
• Direct antiglobulin test on fetal red blood cells with titer
• Positive agglutination in saline or albumin indicate Anti-D
IgM production
Prevention of Rh Alloimmunization
• Anti-D immune globulin/RhIG
– IgG anti-D manufactured from human plasma (primarily
male who undergo repeat injections of Rh+ blood)
– Preparation methods
• HyperRho S/D®, RhoGAM®
– Cohn cold ethanol fractionation followed by viral-clearance
ultrafiltration – intramuscular only as IgA and other plasma
proteins have the potential to produce anaphlyaxis
• Rhophylac®, WinRho SDF®
– Ion-exchange chromatography isolation – intramuscular or IV
Prevention of Rh Alloimmunization
• Anti-D immune globulin
– Mechanism of action: epitope masking?, rapid
clearance?
– Guidelines
• Weak D positive managed as Rh• Mother Rh-, fetus confirmed or suspected Rh+
– 300 micrograms early in third trimester
– 300 micrograms if there is increased risk of feto-maternal
hemorrhage
» Repeat doses if risk is ongoing, guided by titers
– 300 micrograms within 72 hrs after delivery of a Rh+ infant
» If inadvertently not administered, give ASAP – partial
protection has been seen up to 13 days after birth
Diagnosis and Treatment of Intrauterine fetal anemia
• Diagnostic techniques:
–
–
–
–
Ultrasound
Doppler assessment of MCA peak velocity
Percutaneous umbilical cord sampling
Allele-specific PCR on fetal cells in amniotic fluid
• Itrauterine fetal transfusion
– Transfuse when fetal hematocrit falls below 2 standard deviations of mean
hematocrit for gestational age
– Can be performed between 18 and 35 weeks
– Intraperitoneal transfusions not as effective as intravascular transfusions in
hydropic fetus due to congested lymphatics
– ABO type O, Rh- packed RBC utilized
– Fetal loss 1-2% with overall survival of 85% after transfusion
Case Presentation
• Finalizing the case
– What do you order?
• Maternal Anti-D titer
– 1:64
• US of fetus and MCA peak velocity
– Slightly hydroptic fetus
– MCA peak velocity elevated
• Fetal CBC
– Moderate anemia
• Intrauterine fetal transfusion
performed by IR
– Mom transferred to high risk OB
service where last you heard she was
doing very well and expecting to be
discharged
Questions
?