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 versus host disease
1.
2.
3.
4.
5.
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
RECIPIENT CELLS
PLASMA
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 antired 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 PLA1)
• 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
---------------<95
4.0/24/1.1
Ca – 4.9 mg/dL (wnl)
Mg – 2.0 mg/dL (wnl)
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
Cordocentesis
Invasive procedures: genetic amniocentesis;
chorionic villus sampling; multi-fetal reduction; fetal
blood sampling
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
– Antigen positive individuals will not form antibodies
•
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
– 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
Antigens infrequently
associated with severe
HDFN
Jsb
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
?