DR Joe Chaffin Osler Course

The Osler Institute
Blood Bank I
D. Joe Chaffin, MD
Bonfils Blood Center, Denver, CO
The Fun Just Never Ends…

A. Blood Bank I
• Blood Groups
B. Blood Bank II
• Blood Donation and Autologous Blood
• Pretransfusion Testing
C. Blood Bank III
• Component Therapy
D. Blood Bank IV
• Transfusion Complications
* Noninfectious (Transfusion Reactions)
* Infectious (Transfusion-transmitted Diseases)
E. Blood Bank V (not discussed today but available
at www.bbguy.org)
• Hematopoietic Progenitor Cell Transplantation
F. Blood Bank Practical
• Management of specific clinical situations
• Calculations, Antibody ID and no-pressure sample
questions
Blood Bank I
Blood Groups
I. Basic Antigen-Antibody Testing
A. Basic Red Cell-Antibody Interactions
1. Agglutination
a. Clumping of red cells due to antibody coating
b. Main reaction we look for in Blood Banking
c. Two stages:
1) Coating of cells (“sensitization”)
a) Affected by antibody specificity, electrostatic
RBC charge, temperature, amounts of antigen
and antibody
b) Low Ionic Strength Saline (LISS) decreases
repulsive charges between RBCs; tends to
enhance cold antibodies and autoantibodies
c) Polyethylene glycol (PEG) excludes H2O, tends
to enhance warm antibodies and autoantibodies.
2) Formation of bridges
a) Lattice structure formed by antibodies and RBCs
b) IgG isn’t good at this; one antibody arm must
attach to one cell and other arm to the other cell.
c) IgM is better because of its pentameric structure.
P}Chaffin (12/28/11) Blood Bank I page 1
Pathology Review Course
2. Hemolysis
a. Direct lysis of a red cell due to antibody coating
b. Uncommon, but equal to agglutination.
1) Requires complement fixation
2) IgM antibodies do this better than IgG.
B. Tube testing
1. Immediate spin phase
a. Mix serum, 2-5% RBC suspension; spin 15-30 sec.
1) Most common: 2 drops serum, 1 drop RBCs.
b. Antibodies reacting here are usually IgM
2. 37 C phase
a. Add potentiator (+/-), incubate at 37 C, spin.
b. Potentiators and incubation times:
1) 10-15 minutes for LISS
2) 15-30 minutes for albumin or PEG
3) 30-60 minutes for no potentiation
3. Indirect antiglobulin (“antihuman globulin”) phase
a. Wash above to remove unbound globulins.
b. Add antihuman globulin, spin.
C. Alternatives to tube testing
1. Column agglutination technology (Gel testing)
a. Add RBCs and plasma to top of tube, incubate, spin.
b. Microtubes are filled with gel particles and anti-IgG
1) Anti-IgG grabs onto IgG-coated RBCs and inhibits
their migration through gel immunologically
2) Gel particles separate RBC clusters physically
(inhibit agglutinates from migrating through gel).
c. Results:
1) Negative: RBCs form button at bottom of
microtube.
2) Positive: RBCs stopped in areas through the
microtube (more positive = higher position in tube)
d. Can be automated (ProVue machine)
e. Similar sensitivity to PEG tube testing
2. Solid-phase Red Cell Adherence Testing
a. Antibody binds to lysed or intact RBC antigens that
are bound by manufacturer to the sides of microwells
b. Add patient serum, incubate, wash: If positive,
antibody binds to test RBCs.
c. Indicator RBCs (coated with monoclonal anti-IgG)
attach to antibody on test RBCs.
d. Spin and interpret
1) Negative: RBCs in a button at bottom of microwell,
(indicator cells didn’t bind to the test RBCs).
2) Positive: RBCs spread in a “carpet” all along the
microwell (indicator cells did bind to test RBCs).
e. Can be automated (Galileo, Galileo Echo, NEO)
f. Similar sensitivity to PEG tube testing
page 2 Blood Bank I P}Chaffin (12/28/11)

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D. The Antiglobulin Test (“Coombs Test”)
1. Indirect: see above; demonstrates in-vitro RBC coating
with antibody and/or complement.
2. Direct: red cells from patient washed, then mixed with
antihuman globulin; demonstrates in-vivo RBC coating
with antibody and/or complement.
3. IAT variations
a. Unknown antibody check: Use RBCs with a known
antigen profile, as in an antibody screen
b. Unknown RBC antigen check: Use serum with known
antibody specificity, as in RBC antigen testing
c. Can be used to check for an unknown antigen OR
unknown antibody, as in the crossmatch procedure
4. Specificity possibilities for the antiglobulin
a. Anti-IgG, -C3d (“polyspecific”); most common to start
1) Detect red cells coated with either of the above
2) May also detect other immunoglobulins (because
the anti-IgG detects light chains, too)
b. Anti-IgG and anti-IgG (heavy chain-specific)
1) Both detect IgG-coated red cells
2) Anti-IgG used for PEG, gel, and solid phase tests
c. Anti-C3b, -C3d
1) Detects either of the above complement components
2) Most useful in evaluating IgM-related hemolysis,
cold agglutinin disease
5. IgG-sensitized RBCs (“Coomb’s control”, “check cells”)
a. Use after negative DAT or IAT tube test (not gel or
solid-phase) to ensure functioning of AHG reagent
b. Add IgG-coated cells to AHG-cell mixture
c. Negative = bad AHG or no AHG added
d. Other errors (e.g., omitting test serum) missed.
E. Dosage
1. Some antibodies react more strongly with RBC antigens
that have homozygous gene expression.
2. For example, imagine a hypothetical anti-Z
a. Patient 1 genotype: ZZ (Homozygous for Z)
b. Patient 2 genotype: ZY (Heterozygous for Z)

c. If anti-Z shows dosage, it will react stronger with
patient 1’s RBCs (see below).
RBC Genotype Reaction with anti-Z

ZZ 3+
ZY 1+
3. Most common in Kidd, Duffy, Rh and MNS systems
F. Enzymes
1. Proteolytic enzymes (e.g., ficin, papain) cleave RBC
surface glycoproteins and can strengthen reactions by
enhancing antigen expression or allowing antibodies to
bind better to previously shielded antigens
2. Enzymes may also directly destroy other antigens
3. Useful in antibody identification to confirm or refute a
particular antigen as target of an antibody (see table)
4. The “Enzyme Classification”
Enhanced Decreased Unaffected
ABO-related MNS System Kell System
ABO, H Systems Duffy System Diego System
Lewis System Lutheran System Colton System
I System
P System
Rh System
Kidd System
G. Neutralization
1. Certain substances, when mixed with a red cell antibody,
inhibit the activity of that antibody against test red cells.
2. Some of these are pretty weird! (See table below)
Neutralization of Antibodies
ABO Saliva (secretor)
Lewis Saliva (secretor for Leb)
Hydatid cyst fluid
P1
Pigeon egg whites
Sda Human urine
Chido, Rodgers Serum
H. Lectins
1. Seed/plant extracts react with certain RBC antigens
2. Especially useful in polyagglutination (T, Tn, etc)
3. May be commercial or homemade
Lectin Specificity
Dolichos biflorus A1
Ulex europaeus H
Vicia graminea N
Arachis hypogea T
Glycine max T, Tn
Salvia Tn
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II. Blood Groups
A. General characteristics
1. Definition
a. Blood group antigen: Protein, glycoprotein, or
glycolipid on RBCs, detected by an alloantibody
1) NOTE: Antigens are not limited to RBCs
b. Blood group system: Group of blood group antigens
that are genetically linked (30 total systems per ISBT)
2. Significance
a. “Significant” = antibody causes HTRs or HDFN
b. Most significant antibodies are “warm reactive”;
meaning they react best at IAT (37 C).
c. Most insignificant antibodies are “cold reactive”;
meaning they react best below 37 C.
d. Warm antibodies most often IgG, colds usually IgM.
e. IgM antibodies are usually “naturally occurring” (no
transfusion or pregnancy required for their formation).
f. ABO is the exception; see asterisks in table below
“WARM-REACTIVE” “COLD-REACTIVE”
IgG IgM
Require exposure Naturally occurring
Cause HDN No HDN*
Cause HTRs No HTRs*
“Significant” “Insignificant”*
B. ABO and H Systems
1. Basic biochemistry (see figure below)
a. Type 1 and 2 chains
1) Type 1: Glycoproteins and glycolipids in secretions
and plasma carrying free-floating antigens
2) Type 2: Glycolipids and glycoproteins carrying
bound antigens on RBCs.
b. Se gene (FUT2; FUT = “fucosyltransferase”)
1) “Secretor” gene (chrom 19); Precursor to making A
or B antigens in secretions
2) FUT enzyme adds fucose to type 1 chains at
terminal galactose; product is type 1 H antigen
3) 80% gene frequency
c. H gene (FUT1)
1) Closely linked to Se on chrom 19
2) FUT enzyme adds fucose to type 2 chains at
terminal galactose; product is type 2 H antigen.
3) Virtually 100% gene frequency (Bombay = hh).
d. H antigen required before A and/or B can be made on
RBCs (type 2 H) or in secretions (type 1 H).
1) Single sugar added to a type 1 or 2 H antigen chain
makes A or B antigens and eliminates H antigen.
a) Group A sugar: N-acetylgalactosamine

b) Group B sugar: Galactose
2) As more A or B is made, less H remains.

a) H amount: O > A2 > B > A2B > A1 > A1B
2. ABO antigens
a. Genotype determined by three genes on long arm of
chrom 9: A, B and O (O is nonfunctional).
b. A and B genes code for transferase enzymes, not
directly for an antigen (as above)
c. ABO antigens begin to appear on fetal RBCs at 6
weeks gestation; reach adult levels by age 4.
1) Also present on platelets, endothelium, kidney,
heart, lung, bowel, pancreas tissue
3. ABO antibodies
a. Antibodies clinically significant, naturally occurring
b. Begin to appear at 4 months of age; reach adult levels
by age 10 and may fade with advanced age
f. Three antibodies: anti-A, anti-B and anti-A,B; differ
by blood group
1) Group A and B: Anti-A or –B is predominantly
IgM, but each reacts strongly at body temperatures.
2) Group O: Anti-A and –B are predominantly IgG,
and react best at body temperatures
3) Group O: Anti-A,B is IgG reacting against A and/or
B cells (reactivity can’t be separated into individual
specificities).
Type Whites Blacks Asians Native Americans

O 45% 49% 40% 79%
A 40% 27% 28% 16%
B 11% 20% 27% 4%
AB 4% 4% 5% <1%
4. ABO blood groups
a. Group O
1) The most common blood group across racial lines
2) Genotype: OO
3) Antigen: H
a) Ulex europaeus lectin reacts with H antigen.
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4) Antibodies: Anti-A, anti-B, anti-A,B (see above)
a) Because of strong IgG component to all above
antibodies, mild HDFN is common in O moms
b) Why not severe? Weak fetal ABH expression,
soluble ABH antigens (neutralize antibodies)
b. Group A
1) Possible genotypes: AA, AO
2) Antigens: A, H
3) Antibody: anti-B (primarily IgM).
4) A subgroups
a) A1 (80%) and A2 (~20%) most important
b) Monoclonal anti-A agglutinates both types well
c) A1 red cells carry about 5x more A on RBC
surfaces than A2 cells
d) Qualitative differences also exist in the structure
of the antigenic chains (type 3 and 4 for A2).
e) 1-8% of A2 and 25% of A2B form anti-A1.
• Usually clinically insignificant IgM
• Common cause of ABO discrepancies.
• If reactive at 37C, avoid A1 RBC transfusion.
f) Dolichos biflorus lectin agglutinates A1 but not
A2 RBCs.
c. Group B
1) Genotypes: BB, BO
2) Antigens: B, H
3) Antibodies: Anti-A (primarily IgM).
4) B subgroups: Usually unimportant and less frequent
d. Group AB
1) Least frequent ABO blood type (about 4%)
2) Antigens: A and B (very little H)
a) Can be further subdivided into A1B or A2B
depending on the status of the A antigen
3) Antibodies: none
5. ABO testing
Cell Serum ABO
Anti-A Anti-B A1 cells B cells Group
4+ 0 0 4+ A
0 4+ 4+ 0 B
4+ 4+ 0 0 AB
0 0 4+ 4+ O
a. Cell grouping (“forward grouping”)
1) Patient red cells agglutinated by anti-A, anti-B.
b. Serum grouping (“reverse grouping”, “back typing”)
1) Patient serum (or plasma) against A1 and B RBCs.
c. Note the opposite reactions!
1) If forward reactions are not opposite of reverse, an
ABO discrepancy is present.
d. Both serum and cell grouping required unless testing
babies < 4 months of age or reconfirming ABO testing
done on donor blood (requires cell grouping only).
6. ABO discrepancies
a. Disagreement between the interpretations of cell and
serum grouping (e.g., forward = A, reverse = O);
caused by antigen and/or antibody problems or
technical errors.
b. Antigen problems
1) Missing antigens
a) A or B subgroups
b) Transfusion or transplantation
c) Leukemia or other malignancies
2) Unexpected antigens
a) Transfusion/transplantation out-of-group
b) Acquired B phenotype (more below)
c) Recent marrow/stem cell transplant.
d) Polyagglutination
c. Antibody problems
1) Missing antibodies
a) Immunodeficiency
b) Neonates, elderly, or immunocompromised
c) Transplantation or transfusion
d) ABO subgroups
2) Unexpected antibodies
a) Cold antibodies (auto- or allo-)
b) Anti-A1
c) Rouleaux/plasma expanders (false positive)
d) Transfusion or transplantation
e) Reagent-related antibodies
d. Technical errors
1) Sample/reagent prep, mix-ups, or interpretation
errors
7. Weird stuff about ABO
a. Acquired B phenotype
1) A1 RBC contact with enteric gram negatives: Colon
cancer, intestinal obstruction, gram-negative sepsis
2) AB forward (with weak anti-B reactions), A reverse
3) Bacterial enzymes deacetylate group A GalNAc;
remaining galactosamine looks like B and reacts
with forms of monoclonal anti-B (ES-4 clone).
Cell Typing Serum Typing
Anti- Anti- A1 B
Interp Interp
A B cells cells
4+ 1-2+ AB 0 4+ A
4) Use monoclonal anti-B that does NOT recognize
acquired B, acidify serum (no reaction with anti-B)

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b. B(A) phenotype
1) Opposite of acquired B (group B patients with weak
A activity); this condition is inherited, not acquired
2) Cross-reaction with a specific monoclonal anti-A;
test using different anti-A shows the patient as B.
c. Bombay (Oh) phenotype
1) Total lack of H, A and B antigens due to lack of H
and Se genes (genotype: hh, sese)
2) Naturally occurring strong anti-H, anti-A, anti-B
3) Testing: O forward, O reverse, but antibody screen
wildly positive and all units incompatible
4) “Para-Bombay” phenotype
a) Like Bombays, are hh, but unlike Bombays,
have at least one Se gene
b) Phenotypes: Ah, Bh, ABh
c) RBCs may be Bombay-like, but may also show
free or RBC A or B antigens (unless group O).
d) Allo-anti-H present in serum.
5) Both Bombay and Para-Bombay need H-negative
blood (from Bombay donors)
8. Consequences of ABO incompatibility
a. Severe acute hemolytic transfusion reactions
1) Among most common blood bank fatalities
2) Clerical errors
b. Most frequent HDFN; usually mild, however
C. Lewis System
1. Biochemistry (see figure below)
a. Type 1 chains only
b. One gene: Le (FUT3)
1) Second gene, le, is nonfunctional
c. FUT enzyme adds fucose to subterminal GlcNAc
(left side of figure below).
1) This makes Lea (Lewis A) antigen.
2) Lea antigens cannot be modified to make Leb.
d. In secretors, Se product (FUT2) adds fucose, then Le

product adds fucose; this makes Leb (Lewis B).
1) In secretors, Leb formation occurs preferentially.

2) As a result, the vast majority of the chains of those
who carry Le and Se are Leb rather than Lea.
3) In non-secretors, Lea is only possible Lewis antigen.
e. Unlike ABO, antigens are not tightly bound
(remember, they are made from type 1 chains); rather,
they adsorb onto the surface of RBCs.
1) Leb does this better than Lea; another reason that
most adults with both Le and Se will be Le(a-b+).
2) Le(a-b+) people still have Lea, just in much smaller
quantities that may not show up on RBCs.
f. Same chain can carry Le and ABO antigens (unlike the
inverse relationship with ABO and H).
2. Lewis phenotypes, antigens, and antibodies
a. Phenotypes: Le(a-b+), Le(a+b-), Le(a-b-)
b. 22% of blacks are Le(a-b-), vs. only 6% of whites.
c. Antibodies are naturally occurring, cold-reacting IgM.
1) Primarily in Le(a-b-)
2) Neutralize with saliva from secretors.
3) Antibodies commonly also show ABH specificity
(e.g., anti-LebH reacts best with O or A2 RBCs)
3. Consequences of incompatibility
a. Antibodies are generally insignificant
b. Rare HTRs (more commonly with anti-Lea)
c. No HDFN (antibody doesn’t cross placenta and Le
antigens are not present on fetal RBCs).
4. Weird stuff about Lewis
a. Lewis antigens decrease during pregnancy.
1) Pregnant patients may appear Le(a-b-) and have
transient, insignificant Lewis antibodies.
2) Increased plasma volume dilutes the antigens and
increased plasma lipoproteins strip the antigens
b. Le(a-b+) people don’t make anti-Lea.
1) Still have Lea, just not visible on their RBCs.
c. Children’s Lewis type may vary, as antigen chains are
converted [more Lea than Leb initially, with a transient
period of Le(a+b+)]; by age 2, are Le(a-b+)
d. Infection associations:
1) H. pylori attaches to gastric mucosa via Leb antigen.
2) Norwalk virus also attaches via Leb
3) Le(a-b-) are at risk for Candida and E. coli infection
D. I System
1. Antigens built on type 2 chains.
2. Expression is age-dependent.
a. Simple chains found on neonates make i antigen.
b. Branched chains in adults make I antigen.
c. “Big I in big people, little i in little people”
d. Occasional adults lack I; they are known as “iadult”;
more common in Asians

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3. Antibodies (usually autoantibodies)
a. Cold reacting IgM, with auto-anti-I seen commonly
b. Naturally occurring, common, usually insignificant
c. Like Lewis, antibodies commonly have H specificity
as well (e.g., anti-IH reacts better against O and A2)
4. Classic associations
a. Auto-anti-I
1) Cold agglutinin disease
2) Mycoplasma pneumoniae infection
b. Auto-anti-i
1) Associated with infectious mononucleosis
2) Less often a problem than auto-anti-I
c. Iadult phenotype
1) Cataracts
2) HEMPAS
E. P System (the cool one)
1. Also built on ABO-related chains
2. Antigens
a. P1 is the only antigen
1) P, Pk not officially in P system anymore
2) These three antigens define the overall P phenotype.
3) Most common P phenotype: P1 (P+P1+Pk–).
b. Very rare people lack all three and make anti-PP1Pk.
1) Acute HTR and early spontaneous abortions
c. P antigen is parvovirus B19 receptor.
d. Pk antigen is receptor for various bacteria and toxins
3. Antibodies (anti-P1)
a. Cold reacting, naturally occurring, insignificant IgM;
rare anti-P1 reactive at AHG is potentially significant
b. Titers elevated in those with hydatid cyst disease
(Echinococcus) and bird handlers
1) Bird feces contains P1-like substance.
c. Neutralized by hydatid cyst fluid, pigeon egg whites
4. Association with paroxysmal cold hemoglobinuria
a. Biphasic IgG with anti-P (not P1) specificity
1) Binds in cold temps, hemolyzes when warmed
2) “Donath-Landsteiner biphasic hemolysin”
b. Historically in syphilis, now after viral infx in children
F. Rh System
1. Second most important blood group (after ABO)
2. Old (incorrect) Rh antigen terminology systems
a. Fisher-Race (DCE or CDE)
1) Five major antigens: D, C, E, c, e
a) “Rh positive” really means “D positive.”
b) Absence of D designated “d” (no d antigen)
c) C/c and E/e are antithetical (e.g., can’t have both
C and c or E and e from same chromosome)
2) Eight potential combinations based on presence of
genes for above antigens (ie, “DCe”, “dce”, etc.)
b. Wiener (Rh-Hr)
1) Different, archaic names for the five main antigens
2) Believed that main Rh genes (for presence or
absence of D, for C or c and for E or e) inherited as
one genetically linked group, or “haplotype.”
3) Shorthand names to the haplotypes; nomenclature is
still in use and is essential to know (though theory
of how these are inherited has been disproven).
Wiener’s “Haplotypes”
(with DCE Equivalents)
R1: DCe r’ : dCe
R2: DcE r”: dcE
R0: Dce r : dce
Rz: DCE ry : dCE
a) Rules for converting Wiener’s modified
haplotypes into Fisher-Race terminology:
• “R” = D, “r” = d
• “1” or “prime” = C
• “2” or “double prime” = E
• “0” or “blank” = ce
• Any sub- or superscript letter = CE
4) Only four of the above combinations occur with
significant frequency: R1, R2, R0 and r. (~97% of
blacks and whites use only these four).
• R0 most common in blacks, least common in
whites.
• r is always second in frequency.
• R1 always comes before R2.
“The Big Four”
Whites: R1 > r > R2 > R0
Blacks: R0 > r > R1 > R2
5) Asians us. D+; their order is R1 > R2 > r and R0.

c. Current understanting of Rh genetics/structure
1) Two genes, RHD and RHCE (chromosome 1) code
for two main Rh proteins (RHD and RHCE)
2) D type determined by presence/absence of RHD
3) One protein gives both C/c and E/e antigens;
combination determined by which alleles of RHCE
are present (CE, Ce, cE, or ce)
3. Rh antibodies
a. Exposure-requiring, warm-reacting IgG
b. D induces the most antibodies, then c and E
1) Traditional: 80-85% of D negatives make anti-D
when exposed to one unit of D pos RBCs
2) Recent data: 20-30% in hospital settings
c HTRs with extravascular hemolysis

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d Severe and prototypical HDFN with anti-D, severe
HDFN with anti-c, mild HDFN with anti-C, -E, -e
4. Weird stuff about Rh
a. D-negative phenotype
1) Unusual because caused by mutations and deletions
rather than by synthetic actions of a gene product
2) Caucasians: D-negatives have deletion of RHD gene
3) African-Americans: Point mutations in RHD gene
(“pseudogene”)
4) Asians: Usually have inactive RHD gene
b. D Variants
1) Weak D (formerly “Du”)
a) Usual D testing: Monoclonal IgM with
polyclonal IgG read only at immediate spin
b) Almost all D+ test as D+ with these reagents
b) Some D+ individuals have decreased D
expression and require IAT to detect D antigen.
c) Possible reasons for weak D

• Mutated form of RHD
• Point mutation causing altered amino acids
in membrane or inner part of RHD
• Type 1 common in Caucasians
• RHCe on opposite chromosome to RHD (“C
in trans”) inhibits D expression
d) Testing requirements
• Weak D test for all D-negative blood donors
• Not required for D-negative blood recipients
• Previously a concern, for fear of wasting D-
neg units on D+ patients
• Monoclonal antibodies mentioned above
make this very unlikely
• The only patients who definitely need weak
D testing are apparently D-negative babies
with D-negative moms.
e) Weak D moms do not need RhIG prophylaxis
2) Partial D (“D Category”, “D mosaic”)
a) At one time considered a form of weak D
b) Lack portions (epitopes) of D antigen.
c) RHD gene mutations leading to alteration of
exterior part of RHD antigen
d) Antibodies form against absent parts of RHD;
this antibody appears to be anti-D at first glance

e) Classic: Anti-D in a D-positive person
f) Most common: DVI (D “six”) in whites
• Monoclonal anti-D usually types these as D-
negative (prevents D exposure as recipients)
g) Note that partial C and partial e antigens exist,
and can result in unusual antibodies
h) Partial D moms do need RhIG prophylaxis
i) Partial D vs. weak D may be impossible without
molecular testing; if in doubt for prenatal
testing, consider patient D-negative
3) Del (“D-E-L”)
a) Appear D-neg but have tiny amounts of D seen
after elution of reagent anti-D from RBCs
b) Primarily seen in Asian populations (up to 1/3 of
D-negative Asians)
c. These antibodies go together…
1) Anti-E formation commonly accompanied by anti-c
(not necessarily vice-versa)
2) Think “Big 4”; R2R2 gives both E and c exposure
d. Compound Rh antigens
1) G = Antigen present when either C or D is present
• Anti-G reacts against (D+C-), (D-C+), or
(D+C+) RBCs (rarely against D-C-G+)
• Common presentation: D-negative person forms
anti-D when not obviously exposed to D
• Important because if D-neg mom has anti-G, she
DOES still need RhIG to prevent anti-D
• Can cause HTRs (give D-C- blood)
• See bbguy.blogspot.com/2011/08/g-whiz.html
2) f = Present when RHce is inherited (r and R0).
• Anti-f is often seen with anti-e or anti-c
• Can cause mild HDFN and HTR
G. Kidd System
1. Kidd antigens
a. Jka, Jkb, Jk3 (very high frequency)
b. Jka slightly more common than Jkb in African
Americans but similar in whites and Asians
c. Antigens reside on a urea transport protein
2. Kidd antibodies
a. Exposure requiring, warm-reacting IgG (often with
IgM component as well)
1) Can fix complement (with IgM component)
2) Severe acute HTRs possible
b. Marked dosage effect
1) Antibodies may not react at all against cells with
heterozygous Kidd antigens
c. Variable antibody expression
1) Antibody often disappears with time/storage.

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3. Weird stuff about Kidd
a. Delayed HTRs (most famous association)
1) Anamnestic response
2) Intravascular and often severe
b. Mild HDFN at worst
1) Child can only be one antigen different from mom;
remember dosage discussion above.
H. MNS System
1. Basic biochemistry
a. Glycophorin A (GPA) carries M or N antigens.
b. Glycophorin B (GPB) carries S or s, and U antigens.
2. MNS antigens
a. M frequency roughly equals N (each ~75%)
b. s (~90%) is more frequent than S (~50%W, ~30%B)
c. If S-s- (as seen in 2% of African-Americans), may also
be U-negative (U is extremely high frequency).
d. Vicea graminea lectin reacts against N antigens
e. Mur: Hybrid antigen seen in nearly 10% of Chinese
1) Significant antibodies can form; more frequent in
some areas than anything but anti-A or -B
3. MNS antibodies
a. M and N antibodies are mostly opposite of S, s and U
antibodies (see below)
Anti-M & anti-N Anti-S, -s and -U
Naturally occurring Require exposure
Cold IgM Warm IgG
Dosage Minimal dosage
Insignificant Significant
b. Anti-M and anti-N can usually be ignored unless

reactive at 37C; not so with anti-S and anti-s
1) Though anti-M is usually insignificant, it has been
rarely associated with severe HDFN.
c. Effect varies by enzyme, but enzymes generally
decrease all MNS antigens except U
4. Weird stuff about MNS
a. N-like antigen (‘N’)
1) GPB always has terminal 5 amino acid sequence
that matches GPA’s terminal sequence when it is
expressing N; this is known as ‘N’.
a) Not really true N antigen, but it’s close enough
to prevent most M+N- from making anti-N.
2) Seen in all except those who lack glycophorin B.
a) <1% of blacks lack S, s, and U; rare in whites
b) Anti-N nearly exclusive to African-Americans
b. Auto-anti-N induced by hemodialysis
1) Formaldehyde sterilization of machine
2) Modification of N leads to rare autoantibody
I. Duffy System
1. Duffy antigens and genes
a. Fya from Fya gene; high frequency in Asians
b. Fyb from Fyb gene; high frequency in caucasians
c. Absence of both antigens, Fy (a-b-), is most common
Fy phenotype in African-Americans (68%, even
higher in Africa).
1) Due to inheritance of two copies of Fy gene, which
gives no functioning Duffy glycoprotein
2) Fy is an Fyb gene variant, and gives Fyb antigen in
non-RBC tissues
2. Duffy antibodies
a. Anti-Fya more common and significant than anti-Fyb
b. Exposure requiring, warm-reactive IgG
c. Marked dosage and variable expression like Kidd Abs
a. Severe HTRs, usually delayed and extravascular
b. Often mild, occasionally severe HDFN
4. Weird stuff about Duffy
a. Fy(a-b-) and malarial resistance
1) Fy(a-b-) humans are resistant to Plasmodium vivax
and P. knowlesi infection.
J. Kell System
1. Extremely important group clinically and serologically
2. Kell antigens
a. Low frequency: K, also known as “KEL1” (9%
whites, 2% blacks), Jsa, Kpa
b. High frequency: k or “KEL2” (99.8%), Jsb, Kpb
c. Kx antigen: Bound to Kell glycoprotein on the red cell
membrane; required for proper Kell antigen expression
1) Actually a separate blood group (Kx system)
2) When Kell antigens decrease, Kx increases (as in
K0, aka “Kell null”)
3) When Kx decreases (as in “McLeod syndrome”, see
later), Kell antigens decrease, too.
d. Kell system antigens destroyed by thiol reagents (2-
ME, DTT, ZZAP) but not by enzymes alone.
3. Kell antibodies
a. Anti-K
1) Most common non-ABO antibody after anti-D
2) Exposure-requiring, warm reacting IgG1
3) More common from transfusion than pregnancy
b. Anti-k
1) Very uncommon due to high antigen frequency
2) Antibody is just like anti-K
a. Severe HTRs
1) May be acute or delayed; usually extravascular.
The Osler Institute
b. Severe HDFN
1) Less common than ABO or RHD HDFN
2) Damages EARLY RBC precursors, so tends to be
suppressive rather than hemolytic
a) Lower bilirubin and reticulocytopenia than with
anti-D HDFN
5. Weird stuff about Kell
a. Kell null phenotype (“K0”)
1) All Kell antigens decreased, Kx increased
2) Significant anti-Ku (“universal”) with exposure
b. McLeod phenotype
1) Kx absent, all Kell antigens markedly decreased
2) No anti-Ku like K0, but can form anti-Kx and anti-
Km (Kell “McLeod”); only compatible with
McLeod RBCs
3) Phenotype is part of McLeod “syndrome”
a) Hemolytic anemia with acanthocytes
b) Myopathy, ataxia, peripheral neuropathy,
cognitive impairment, cardiomyopathy
c) Occasional association with X-linked chronic
granulomatous disease
• NADPH oxidase deficit
• Organisms phagocytized but not killed
• Catalase-positive organisms (Staph)
K. Diego System
1. Over 20 antigen system built on “band 3”
a. Important RBC membrane structure
b. Carries HCO3- anions out of RBCs (for CO2 removal),
and anchors membrane to cytoskeleton
2. Diego antigens
a. Dia and Dib antithetical pair
1) Dia very low frequency except in some South
Americans and Asians
2) Dib very high frequency in all populations
b. Wra and Wrb antithetical pair
1) Wr = “Wright”
2) Wra very low frequency, Wrb very high frequency
3. Diego antibodies
a. Di antibodies are IgG, while Wr antibodies may have
IgM component
b. Both anti-Dia and –Dib can cause HDFN that may be
severe but generally not HTRs
c. Anti-Dib can show marked dosage effect
d. Anti-Wra is common, naturally occurring, and may
cause both HTRs and severe HDFN (IgG + IgM)
e. Anti-Wrb, on the other hand, is rarely seen as an
alloantibody but may be an autoantibody in
autoimmune hemolytic anemia (AIHA)

L. A few other systems and antigens (in brief)
1. Dombrock System
a. Doa/Dob antigens; Dob more frequent
1) Either antibody may cause HTRs but generally
don’t cause HDFN
2) Warm-reactive IgG
b. High frequency antigens Joa, Gya, Hy
1) Mild HTRs or HDFN possible, but antibodies are
very rare
2) Near 100% incidence for all of these
3) Joa- and Hy negative exclusively in blacks
4) Gya negative in Japanese and eastern Europeans
2. Colton (Co) System
a. Antigens (Coa and Cob) located on water transport
membrane protein (aquaporin 1)
b. Coa very high frequency (near 100%), Cob about 10%
c. Both antibodies may cause significant HDFN
3. Lutheran (Lu) System
a. Lua (low frequency; 5-8%) and Lub (very high
frequency; 99.8%) antigens
b. Antibodies uncommon, may be naturally occurring
(anti-Lua), and not usually significant
c. Most enzymes decrease Lu antigen activity.
4. Xg System
a. Gene carried on X chromosome (“X-linked”)
1) Seen in 66% of males and 90% of females
b. Antibody insignificant
5. Yt System
a. Formerly “Cartwright”
b. Yta (very high frequency; 99.8%), Ytb (8%)
c. Antibodies are IgG but not usually significant
(occasional anti-Yta can cause HTRs, however)
6. Vel Antigen
a. Extremely high frequency antigen (>99% in all
populations)
b. Antibody is mix of IgG and IgM
1) May cause severe HTRs and HDFN
2) May interfere with ABO typing due to reaction at
room temperatures
3) May be allo- or autoantibody
7. Landsteiner-Wiener (LW) System
a. LWa antigen is more abundant on D-positive RBCs
b. LW antigens were originally thought to be Rh antigens
c. Antibodies are not generally significant
8. Sda (“Sid”) antigen
a. High frequency (96%)
b. Refractile, small immune complexes with naturally
occurring IgM

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a
c. Lectin of Dolichos biflorus agglutinates Sd positive
RBCs (like A1)
d. Neutralize with guinea pig or human Sda+ urine!
9. Antibodies with “high titer, low avidity” (HTLA) features
a. High frequency antigens that are generally clinically
benign (no HTRs or HDN)
b. Chido, Rodgers most frequent
1) Complement components (C4)
c. Multiple others known
1) Knops (Kna), McCoy (McCa), JMH
d. Must be careful, because some antibodies with similar
features may be significant (anti-Vel, anti-Yta)

The Osler Institute
Blood Bank II
D. Joe Chaffin, MD
Cedars-Sinai Medical Center, Los Angeles, CA
Blood Donation & Pretransfusion Testing

I. Blood Donation
A. Allogeneic (homologous) whole blood donation
1. Process tightly regulated by FDA and AABB
2. Donor screening by history
a. FDA-recognized AABB template called “donor
history questionnaire” (DHQ); see end of this handout
b. Necessary information
1) Full name (generally, formal ID required)
2) Home and/or work address
3) Date of birth/age to make sure over minimum age
a) 17 unless age 16 donors approved by state
4) Reasons for previous deferral
5) Date of last donation
a) > 8 weeks for whole blood
b) > 16 weeks for double red cell collections
c) > 4 weeks for infrequent plasmapheresis
d) > 2 days for single platelet apheresis procedures
e) > 7 days for double/triple platelet apheresis
c. HIV information presented to donor
1) Signs/symptoms and risk factors for HIV
2) Statement: Do not to donate if have any risk factors
or if just wanting HIV test
d. Medication list; prevents donations from those taking:
1) Medications with teratogenic potential
2) Medications with infectious risk
3) Medications that damage platelets
4) See list on page 5
e. Informed consent must spell out risks
1) Not standardized; individual centers must develop
2) Notification that other agencies might be notified of
results (e.g., state and/or local health departments)
3) Statement that blood might not be tested (lab
accidents/errors, breakage, etc.)
f. Donor questions
1) Version 1.3 of DHQ (recognized by FDA in May
2010) contains 48 standardized questions
2) May modify times only to make more restrictive
3) Most omit 2 HIV group O questions (see later)
4) May add additional questions at end only
a) These can be more restrictive only; never less
b) Commonly added questions are about
medications/health issues not covered in DHQ
P}Chaffin (2/26/2014) Blood Bank II page 1

g. Deferrals based on history
1) Permanent/indefinite deferrals
Infectious Risks
-High-risk behavior for AIDS
(IVDA, male-male sexual contact since 1977)
-Receiving money or drugs for sex
-Serologic positive for HIV, HBV, HCV, HTLV
-Viral hepatitis (any) after 11th birthday
-Transfusion of clotting factor concentrates (in hemophilia)
-History of Babesiosis or Chagas’ disease
-Growth hormone from human sources (pre-1985)
-Insulin from bovine sources
-Dura mater graft
Malignancies (see below)
-Leukemia or lymphoma
Teratogens
-Taking etretinate (Tegison)
2) Three year deferrals
Infectious Risks
-Recovered from malaria
-Immigrants from malaria-endemic countries
(after 5 consecutive years of living there)
Teratogens
-Taking acitretin (Soriatane)
3) One year deferrals
Infectious Risks
-Needle sticks or other contact with blood
-Sex contact with person with HIV or hepatitis
-Sex contact with person who used needles for drugs
-Rape victims
-Incarcerated > 72 consecutive hours
-Paying money/drugs for sex
-Blood transfusion (allogeneic); including plasma/clotting
factors in nonhemophiliacs
-Allogeneic transplant of organ/skin/bone
-Living with person with active hepatitis
(exception: Asymptomatic Hepatitis C)
-Receiving Hepatitis B Immune Globulin (HBIG)
-Tattoos/piercings (unless by regulated entity)
-Travel to malaria-endemic areas for residents of non-endemic
countries (>24 hrs, < 5 years)
-Diagnosed with syphilis or gonorrhea
-Non-prophylactic rabies vaccination
-“Travel” to Iraq
page 2 Blood Bank II P}Chaffin (2/26/2014)

The Osler Institute
4) Special situations
a) Malaria (updated to 08/13 FDA Guidance)
• “Endemic area” means area that CDC
recommends chemoprophylaxis to travelers
• “Endemic country” means any country with at
least one endemic area
• Deferrals:
Situation Deferral
History of malaria 3 years (if asymptomatic)
Living in endemic country for more than 5 3 years (if asymptomatic)
consecutive years
Travel by residents of non-endemic countries
Travel to endemic area for more than 24 1 year (if asymptomatic)
hours and less than 5 years
Travel in endemic country but not endemic No deferral
area for more than 24 hours
Travel by previous residents of endemic countries
Travel to endemic area (prior resident of 3 years (if asymptomatic)
endemic country <3 years after leaving)
Travel to endemic area (prior resident of 1 year (if asymptomatic)
endemic country >3 years after leaving)
b) Malignancy deferrals
• Medical director discretion (not mandated)
• Studies do not show that malignancy can be
transmitted via transfusion
• Most defer leukemia/lymphoma permanently,
but some accept cured childhood leukemics
• For solid tumors, most defer indefinitely and
consider re-entry after 1-5 years disease-free
• BCC, localized skin SCC = no deferral.
c) Heart and lung disease
• No specific mandated deferrals
• FDA: Donor free of acute lung disease
• AABB: Donor should be free of heart/lung
disease unless determined suitable by medical
director
• Medical directors determine acceptability
(time since diagnosis, presence of limitations
on activities, proper medical follow-up)
d) Pregnant women: Defer until 6 wks postpartum.
e) Non-routine dental work: Defer for 72 hours.
f) Questions 46, 47: Africa questions
• These questions are omitted since all centers
use anti-HIV test sensitive for HIV group O
• No reported US cases since 1996
• Questions ask about being born, living in,
traveling to, transfused in, or sexual contact
with someone born or living in countries in
Africa since 1977

5) Defer permanently for vCJD risk all donors who:
a) Lived over 3 months in UK, 1980-1996
b) Lived Continental Europe > 5 years, 1980-now
c) Were transfused in the UK, 1980-now
d) Received dura mater transplant, pituitary growth
hormone injections, or bovine insulin injection
e) Have family history of CJD or vCJD
f) Were military members/dependents:
• Stationed at Northern Europe bases
(Germany, UK, Belgium, Netherlands) for 6
months from 1980-1990
• Stationed at other Europe bases (Greece,
Turkey, Spain, Portugal, Italy) for 6 months
from 1980-1996
6) Immunizations
a) General rule: no deferral for killed, toxoid, or
recombinant/synthetic vaccines
b) Live attenuated vaccines (either viral or
bacterial) give deferrals of varying lengths
Immunization Deferrals
Four Weeks: Rubella
Varicella
Two Weeks: Measles
Mumps
Oral polio
Yellow fever
Oral typhoid
No Deferral: Anthrax
Cholera
DPT
Hepatitis A
Hepatitis B
Influenza
Lyme disease
Meningococcus
Pneumococcus
Polio (injection)
RMSF
Typhoid (injection)
12 Months: Unlicensed vaccines
c) Smallpox vaccination
i) Deferrals based on presence/ absence of
vaccine scab and post-vaccination symptoms
ii) No symptoms: defer until scab falls off or 21
days, whichever is longer
iii) With symptoms: defer until 14 days after
symptoms resolve

The Osler Institute
7) Drugs
a) DHQ only requires questioning about a limited
number of medications
i) Some facilities add more to protect donor
b) DHQ V 1.3 drug deferrals for teratogenicity:
i) Etretinate (Tegison): Permanent deferral
ii) Acitretin (Soriatane): Three year deferral
iii) Isotretinoin (Accutaine, Absorica,
Amnesteem, Claravis, Sotret): 30 day
deferral
iv) Finasteride (Proscar, Propecia): 30 days
v) Dutasteride (Avodart, Jalyn): 6 months
c) DHQ V 1.3 drug deferrals for infection risk:
i) Human pituitary growth hormone: Permanent
ii) Bovine insulin: Permanent
iii) Hep B Immune Globulin (HBIG): 1 year
iv) Unlicensed vaccine: 1 year
d) Aspirin/aspirin like meds for platelets (48 hours)
e) Platelet drug deferrals:
i) Piroxicam (Feldene): 48 hours for platelet
donors only
ii) Clopidogrel (Plavix) and ticlopidine (Ticlid):
2 weeks for platelet donors only
f) Warfarin is not addressed in DHQ, but most
defer donors 7 days
g) New drugs are fast arriving
i) Antiplatelet and anticoagulant drugs seen in
donors (e.g., Brilinta, Pradaxa, Xarelto)
ii) Medical director discretion, but most are
deferring like warfarin
3. Donor screening by physical criteria
a. General appearance
b. Arm check
1) Both arms for evidence of IVDA and venous access
c. Physical requirements (weight, BP, pulse are not
standards but most common practice):
Weight: > 110 lbs (50 Kg)

Temperature: < 99.5o F (37.5 C)
Pulse: 50-100 (unless athlete)
Blood pressure: < 180/100
HGB or HCT: > 12.5 g/dL or 38%
4. Donation specifics
a. Amount drawn
1) Maximum of 10.5 ml/Kg is allowed by AABB
2) 500 +/- 50 mL (+/– 10%) most common
a) 450 mL bag also used; limit is 450 +/- 45 mL

b. Time limit
1) < 10 minutes best, but no upper limit defined
a) Beyond 15 minutes, plasma/PLTs not made
5. Testing donor blood (Collection center)
a. ABO grouping
b. RhD typing
1) Weak D required if D negative (see BBI)
c. Antibody detection (“screen”)
1) Unexpected (non-ABO) antibodies in donor serum
2) AABB Standards: If positive, may still use blood,
but only to make products with minimal plasma
(i.e., RBCs ok; can’t make FFP, cryo, or platelets).
3) Label must reflect any positive results that are
identified as clinically significant antibodies.
4) Reality: Most hospitals don’t want this blood
d. Infectious disease screening (as of 2/2014); see
appendix and further details starting on page 13
1) Hepatitis B Tests
a) HbsAg
b) Anti-HBc
c) HBV nucleic acid test (HBV NAT)
2) Hepatitis C Tests:
a) Anti-HCV
4) HCV NAT
3) HIV tests:
a) Anti-HIV-1/2
b) HIV-1 NAT
4) Other tests:
a) Anti-HTLV-I/II
b) West Nile virus NAT
c) Anti-Trypanosoma cruzi (Chagas’ disease)
d) Serologic test for syphilis
6. Testing donor blood (Transfusion Service)
a. Requires confirmation of collection center’s work
b. Confirmatory tests:
1) ABO grouping (RBC grouping only)
2) RhD-negative confirmation
a) Direct testing only of units labeled as D–
b) Weak D testing not required (done already)
c) Units labeled as D+ do not require confirmation
3) Antibody screen and infectious disease screening on
donor units do not require confirmation
B. Donor reactions
1. Vasovagal reactions
a. Most common reaction (2.5% of healthy donors)
1) Most common in young, first-time female donors
2) Can be seen in any donor, though
3) Can happen before, during, or after donation

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b. Signs/symptoms
1) Bradycardia (or at least, non-tachycardia) with
hypotension
2) Syncope
3) Nausea/vomiting
4) Incontinence (uncommon)
c. Treatment is supportive
1) Distinguish from hypotensive shock
2) Elevate feet, cold compresses on neck
2. Hypovolemic shock
a. Extremely uncommon
b. Signs/symptoms
1) Tachycardia with hypotension
2) Loss of consciousness
3) Shock parameters
c. Treatment
1) IV fluids, possibly return blood?
3. Hyperventilation
a. Especially common in first-time donors and children
b. Signs/symptoms
1) Shortness of breath
2) Facial twitching
3) Seizure activity (unusual)
c. Treat with rebreathing (the paper bag thing)
4. Local injuries
a. Hematomas: 0.35% (bruises almost 25%)
b. Nerve injury: less common than bruises (0.9%);
usually resolve on their own
C. Apheresis procedures
1. Separation of blood into components using several
technologies
a. Centrifugation used for donor procedures (others for
therapeutic procedures; see BB Practical section)
b. Separation based on varying density of blood
components
c. Blood drawn into spinning bowl or chamber,
separated, component harvested (all else returned).
2. Targets for donor collection
a. Platelets
b. Plasma
c. Red blood cells
d. Granulocytes
e. Hematopoietic stem cells (after mobilization)
3. Multiple product collections in one procedure possible
a. Common to get single/double/triple apheresis unit with
concurrent plasma collection
b. May also add an RBC collection
c. “Double” RBC collections (no other products)

4. Donor requirements
a. Platelet donors
1) Same hemoglobin/hematocrit requirements as
regular donors
2) Donation interval at least 2 days for single product
(7 days for double/triple/quad)
a) No more than 2 procedures per week
b) No more than 24 procedures per year
c) > 8 weeks after whole blood donation or if
RBCs not returned (unless equipment has less
than 100 mL extracorporeal volume)
d) Physician may waive above if for a particular
patient need (requires written certification).
3) Donation interval 7 days after double/triple products
4) No aspirin/aspirin-effect products in last 48 hrs
5) No clopidogrel or ticlopidine in last 14 days
6) Pre-procedure platelet count > 150,000/L (may use
previous count if not available)
7) Plasma volume loss as per manufacturer
b. Plasma donors
1) “Infrequent” (> every 4 weeks): = whole blood
2) “Serial” (< every 4 weeks):
a) Total protein and SPEP check every 4 months
b) Interval > 48 hours; < 2 collections per 7 days
c) Red cell loss < 25 mL/week, < 200 mL/8 weeks
c. Double red cell donors
1) FDA: “Assure donor safety”
2) Equipment makers have donor requirements that are
OK’ed by FDA when machine is FDA-cleared
3) Double red cell donors are deferred for 16 weeks
d. Multiple products collected at once
1) FDA-cleared equipment specific donor limits
5. Apheresis donor reactions
a. “Citrate effect”
1) Citrate anticoagulant binds free calcium
2) Perioral tingling
3) Tetany and arrhythmias uncommon
4) Slow rate of infusion, give oral calcium
b. Hypersensitivity reactions
1) Classic: Hydroxyethyl starch in WBC donors
a) Given to facilitate better separation of WBCs
from RBCs by inducing RBC aggregation
b) Occasional hypersensitivity reactions seen
II. Autologous Blood Collection
A. Preoperative autologous blood donation (PAD)
1. Less screening stringency than allogeneic collections
2. AABB Standards: Don’t “cross over” units into regular
inventory unless exceptional circumstances

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3. More lenient physical criteria (see table below)
4. Testing regulations
a. Infectious disease screening not required unless units
are to be shipped to another facility
1) Hospital centers collecting auto units for transfusion
are not required to screen those units
2) If not tested, label units “NOT TESTED”
b. Only first donation in a 30-day period MUST be tested
1) Others after that may be labeled “DONOR
TESTED WITHIN THE LAST 30 DAYS”
Parameter Allogeneic Autologous
Donation Interval 8 weeks 72 hours
HB/HCT > 12.5 or 38% > 11 or 33%
Weight > 110 lbs (50 Kg) None
Age > 16 or 17 (varies) None
Infectious Disease Not required unless
Required
Screening shipped
History of Disease
Not eligible Potentially eligible
or Positive Test
5. Potential issues with autologous donations
a. Donor reactions
1) More complex donors with more health problems,
so more likelihood of donor complications
2) Safety of donor during donation is responsibility of
donor center medical director!
b. Clerical errors/transfusion errors
1) Risk of wrong unit going to wrong patient still
present with autologous donations
2) CAP survey (1992): about 1% of hospitals admitted
making transfusion errors with autologous blood
3) Systems must be in place to prevent allogeneic units
from being transfused before autologous units
c. Bacterial contamination
1) Currently a greater risk than HIV or HCV
2) Risk of undetected infection leading to
contamination of unit is not changed by PAD
d. Cost
1) More costly to patients if transfused and hospitals if
not transfused (wastage)
e. Timing
1) Collections should be completed at least 72 hours
before surgery (preferably sooner)
2) Surgery cancellations, etc, can lead to problems
(freeze units? Let them expire?)
f. Positive infectious disease testing
1) If donor has a reactive test, donor and the requesting
physician must be notified

2) Autologous collections with reactive testing should
lead to deferral from future allogeneic donations
B. Acute Normovolemic Hemodilution (ANH)
1. Primary goal: Reduce RBC volume during surgery in
order to prevent exposure to allogeneic blood
a. Studies to date: May work ok, but only with aggressive
hemodilution
b. Can save exposure, but isn’t very cost effective
2. Added benefit: Reduce plasma and platelet needs
3. Remove 1 L or more of blood immediately before surgery
a. Collection is done into multiple standard blood bags
and each bag should be monitored for overfill
b. Standard formulae exist for collection amount;
typically take patients down to HCT 25-28% or so
1) V = EBV x (Hi-Hf)/Hav (source: Waters JH,
“Perioperative Blood Sequestration”, AABB 2008)
2) Key:
i) V = volume to remove
ii) EBV = estimated blood volume
iii) Hi = initial hematocrit
iv) Hf = final hematocrit
v) Hav = average hematocrit during process
c. Shelf life:
1) 8 hours at room temperature
2) 24 hours in monitored refrigerator
4. Replace volume with saline/crystalloid (3 ml per 1 ml of
blood removed) or colloid (1 ml per ml of blood removed)
a. Replace the volume unit by unit; i.e., when a unit is
withdrawn, immediately replace volume (don’t remove
a bunch of blood and THEN correct the volume)
5. Re-infuse blood near end of surgery.
a. Units usually re-infused in reverse order to how drawn
(i.e., last drawn is first re-infused)
b. If using intraoperative salvage, infuse those units first
6. Indications (not standardized)
a. At least 1 L blood loss anticipated in surgery
b. Hemoglobin at least 12 g/dL
c. No active cardiac or other serious medical disease
d. No infection or bacteremia
7. Potential advantages
a. Bleeding more dilute blood leads to less overall
hemoglobin loss (not as huge a benefit as predicted).
b. Decreased blood viscosity increases cardiac output and
may improve oxygenation.
c. Coag factors and platelets survive well for short
periods and help hemostasis.
8. Potential disadvantages
a. Requires training of phlebotomist (most commonly
done by anesthesiologist)

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b. Units should be labeled with full name, date/time,
medical record number, and “FOR AUTOLOGOUS
USE ONLY.”
9. Component sequestration:
a. Similar in thought to ANH, but involves harvesting
specific components (platelets, plasma, RBCs) for
targeted transfusion depending on patient needs
b. More complex and requires lots of expertise
C. Intraoperative blood salvage (“Cell Saver”)
1. Semi-automated process; usually involves processing and
concentrating shed blood from suction apparatus
2. Generally indicated for major surgeries with large
expected blood losses (cardiac, orthopedics, vascular)
a. Requires coordination with team performing recovery
b. Trend: More aggressive use in lower-risk cases
3. Shelf life:
a. 4 hours at room temperature
b. 24 hours in monitored refrigerator
4. Potential problems
a. Air or amniotic fluid embolus risk
b. Hemolysis of processed blood from excessive suction
in the operative field
c. Coagulation factor activation
5. Historical contraindications; Not uniformly used
a. Bacterial contamination of operative field
b. Malignant cells in field
c. Other contaminants in field (cement, irrigation,
amniotic fluid, etc)
D. Postoperative blood salvage
1. Blood reinfused from operative drains with or without
processing (Editorial comment: That is just gross!)
a. Microaggregate filters used during re-infusion
2. Shelf life: 6 hours at room temperature
III. Pretransfusion Testing
A. Basic outline (see figure above)

1. Requires actions by supplier, phlebotomists, transfusion
services, and infusing staff
2. Crossmatch is final check of everyone’s work
a. Main reason for crossmatch: ABO compatibility!
B. Testing recipient blood
1. Request forms
a. Identification critical
1) Number one cause of fatal HTR’s: clerical error!
b. No identification labeling errors are acceptable.
1) Transfusion 1997 37; 1169-72: Mislabeled
specimens 40X more likely to have a blood
grouping discrepancy!
c. Should tell what’s needed and when needed, ordering
provider, and any modifications (washing, irradiation,
etc.) needed
2. Specimen
a. Serum or plasma (red top vs lavender top)
1) Either ok, but non-tube technologies prefer plasma.
b. Required q 3 days with transfusion or pregnancy in
the last three months
c. Retained 7 days after transfusion in the blood bank.
3. Type and hold (T&H)
a. ABO/Rh check only
b. “Hold” means to hold sample, not units.
c. Uncommonly used or even offered now
4. Type and screen (T&S)
a. Includes:
1) Records check
a) Previous antibodies or compatibility problems
b) Not to be used to determine current ABO/Rh,
but should be compared to current results.
2) ABO testing
a) Forward and reverse grouping
b) Resolve any discrepancies
3) Rh typing
a) No weak D test required if D negative.
• Common exception: obstetric patients
4) Antibody screen
a) “Unexpected” (non-ABO) antibody check
b) Panel of 2, 3, or 4 RBC-phenotyped donors
i) Always group O
ii) Most common combination:
• Cell I: R1R1
• Cell II: R2R2
• Cell III: rr (cell III not used with gel)
c) Antigens represented required by FDA:
i) D, C, c, E, e, Fya, Fyb, Jka, Jkb, K, k, Lea, Leb,
M, N, P1, S, s
d) IAT phase is required, IS/37 C are not required.
e) If positive, identify antibody (see BB Practical)
5. Type and crossmatch (T&C)
a. Everything in T&S + crossmatch

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b. Crossmatch types
1) Major crossmatch (or just “crossmatch”)
a) Recipient serum vs donor RBCs
b) Final check of ABO compatibility
c) IAT/AHG phase (“full crossmatch”) not required
if antibody screen is negative
d) Required if ≥ 2 ml of RBCs in product.
2) Minor crossmatch (not required or routinely done)
a) Donor serum vs. recipient RBCs
3) Units go back into inventory if not used
6. Converting a T&S to a T&C
a. ABO check only required (if antibody screen negative)
1) Immediate spin crossmatch
a) Donor RBCs/recipient serum centrifuged at
room temp
2) Computer crossmatch (see below)
7. Variations to the above
a. Infants less than four months of age (neonatal period)
1) Baby’s antibodies = mom’s IgG antibodies.
2) Initial testing:
a) Infant’s ABO (red cell grouping only)
b) Infant Rh type
c) Antibody screen on mom’s or baby’s serum.
3) If screen neg, repeat initial testing and crossmatch
not needed in same hospital stay up until age 4
months
4) Serum grouping not required unless giving non-
group O RBCs
b. Electronic (computer) crossmatch
1) Computer system verifies ABO compatibility
between donor and recipient (replaces immediate
spin crossmatch)
2) Requires a patient with a negative antibody screen
both now and in the past
3) Requires two separate ABO determinations of the
patient (either on different specimens or repeated on
the same specimen with different reagents)
4) Requires a properly validated computer system
8. Reasons for positive major crossmatch
a. With positive antibody screen
1) Alloantibodies
2) Autoantibodies
3) Reagent antibodies
4) Rouleaux and other false positives
b. With negative antibody screen
1) ABO incompatibility
2) Antibodies vs low-incidence antigens
3) Positive donor DAT

9. Routine blood order templates
a. Maximum Surgical Blood Ordering Schedule
(MSBOS), or just “routine surgical blood orders”
b. Multidisciplinary and institution-specific
c. Gives surgeons / blood bankers a guide to how many
RBC units to crossmatch (or not) for a given procedure
IV. Transfusion-transmitted Diseases (See April
2011 Podcast)
A. Current risk of disease transmission
1. For perspective, risk of acute hemolytic reaction stated as
1 in 25,000 transfusions
2. Risk of dying in the hospital from something other than
transfusion problem: 6 per 1000!
Organism Current Risk Estimate
HIV-1 1 in 1,467,000
Hepatitis B 1 in 765,000-1,000,000
Hepatitis C 1 in 1,149,000
HTLV-I 1 in 4,364,000
HIV-2 Remote
WNV Remote
Syphilis Remote
T. cruzi Unknown, likely remote
Bacteria 1 in 75,000 platelet transfusions
1 in 500,000 RBC transfusions
B. Hepatitis viruses
1. Hepatitis A virus
a. Fecal-oral transmission (30 day incubation)
b. Generally not a big blood banking problem (not tested)
c. Some concern in pooled products
1) Solvent-detergent treatment doesn’t deactivate
HAV (nonenveloped).
2) Transmission possible in pooled factor concentrates
d. Not prone to chronicity like HBV and especially HCV
2. Hepatitis B virus
a. DNA virus (Hepadnavirus)
b. Blood transmission, intimate contact less likely
c. Both cellular and plasma components transmit.
d. Incubation period: approximately 8-12 weeks
e. Clinical
1) Primary infection may be subclinical (65%) or only
mild (jaundice, nausea, fatigue, dark urine).
2) Fulminant presentation rare
3) Chronic infection (“carrier state”) much less likely
than with HCV (< 5% of adult infections)
a) Greatly decreased carriers since routine
vaccination
b) 400 million worldwide carriers, per WHO

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f. Current testing (see appendix)
1) Anti-HBc EIA/ChLIA and HBsAg EIA/ChLIA
a) Confirmatory test for HBsAg: neutralization
b) No confirmatory test for anti-HBc
2) HBV NAT now required (done in combination with
HIV and HCV)
3) No anti-HBsAg testing (vaccinated donors positive)
4) HBV is currently the most likely of the major
viruses to be transmitted via transfusion!
g. Serology patterns below
HBV ANTI-HBc ANTI-HBc ANTI-
HBsAg INTERPRETATION
DNA (TOTAL) (IGM) HBsAg
– – – – – Incubation
+ + + + – Acute infection
– – + – + Recovered infection
+/– + + – – Carrier
– – – – + Vaccinated
Probable false pos.,possible
– – + – – early or chronic infection
h. Deferrals with HBV NAT (source: FDA Guidance
2012):
3. Hepatitis C virus
a. RNA virus
b. 0.5-1.0% of US blood donors
c. Both cellular and plasma components transmit.
d. Strong association with chronic hepatitis (75%),
cirrhosis, and hepatocellular carcinoma (>HBV)
1) Currently #1 reason for hepatic transplant in the US.
2) Initial presentation mild or asymptomatic
e. Donor testing (see appendix)
1) Antibody test is anti-HCV (EIA/ChLIA)
a) Window period with antibody test: 70-80 days

b) During some of this time, HCV RNA is
detectable by PCR testing, and the virus is
transmissible by transfusion (see below).
c) Confirmation:
i) Reactive anti-HCV historically confirmed by
Recombinant ImmunoBlot Assay (RIBA);
NOT AVAILABLE
ii) Instead, confirm either with a different EIA
(requires FDA variance) OR with a specific
HCV NAT licensed for supplemental testing
2) Nucleic acid testing (HCV NAT)
a) Window period from 70-80 days to about 7 days
b) Typically done in combination with HBV and
HIV NAT
3) See appendix for deferral guidelines
4. Other hepatitis viruses we don’t test for:
a. Hepatitis D virus
1) Formerly known as “delta agent”
2) Blood transmission
3) “Defective” virus (requires coating with HBsAg in
order to cause disease).
b. Hepatitis E virus
1) Fecal-oral transmission
2) Epidemics in India and Asia; rare transfusion
transmission
C. Retroviruses
1. HIV-1 and HIV-2
a. RNA retrovirus identified in 1984
1) Hemophiliacs and homosexual men first
2) Transfusion, sexual contact, breast-feeding, blood
b. Clinical/pathophysiology
1) Symptoms in acute infection: “flu-like”
2) LONG asymptomatic period (often over ten years),
then rapid immune compromise
3) Damage caused by attack on CD4+ lymphocytes
4) Death secondary to opportunistic infections or
malignancies like Kaposi’s or CNS lymphoma
c. Testing
1) Antibody testing
a) Required since 1985
b) Window period = 20-22 days
2) Organism testing
a) HIV-1 antigen (p24) testing March 1996
• Reduced window period to about 16 days
b) p24 testing replaced in 1999-2000 by nucleic
acid testing for HIV RNA (HIV NAT)
• Reduction of window period to 9-10 days
d. Both cellular and plasma products can transmit HIV-1
e. See appendix for deferral guidelines

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f. HIV-2
1) Related virus found originally in West Africa
2) Really, really rare
3) Less readily transmitted vs HIV-1, with less AIDS
4) No licensed confirmatory test
2. HTLV I/II
a. Transmission through cellular products only
b. HTLV-I disease associations
1) Adult T-cell leukemia/lymphoma (ATLL)
2) HTLV-associated myelopathy (HAM; formerly
called “tropical spastic paraparesis”)
c. HTLV-II: no clear-cut disease associations
d. Both transmitted readily, but actual post-transfusion
disease is very unlikely (~99% of infected no disease)
e. See testing discussion in appendix
D. Other organisms for which we test:
1. West Nile virus (WNV)
a. RNA virus causing disease in birds; humans incidental
(most flu-like, some meningitis/encephalitis)
b. 2012 large increase in cases in US
c. Testing done via pooled NAT (until high risk of
disease in area, then single donor)
d. Deferrals:
1) Confirmed/suspected WNV infection: 28 days from
symptom onset or 14 days after symptoms resolved
2) Positive test only: 120 days from test date
2. Trypanosoma cruzii (Chagas’ disease)
a. Transmitted through bite of reduviid bug (“kissing
bug”) in Central/South America
b. Potentially growing problem with immigration
(roughly 1 in 20,000 donors, higher in immigrants)
c. Specific question on donor questionnaire (permanent
deferral for history of Chagas’ disease); the problem is
that many are asymptomatic.
d. Screening test (EIA) required as of 2011
1) Testing allowed to be one time per lifetime of donor
3. Treponema pallidum
a. Organism doesn’t survive well in refrigerated storage
(48-96 hours); not considered a large problem
b. Surrogate marker for high-risk behavior
c. See testing discussion in appendix
4. Cytomegalovirus (CMV)
a. Extremely common DNA virus (> 50% are exposed)
that lives in WBCs only (monocytes).
b. Causes severe infections in immunocompromised
adults and neonates, but minimal disease in healthy
people (may have cold-like symptoms)
c. Prevent with seronegative donors and leukocyte
reduced products (see discussion in BB III)

d. Testing not required but available
5. Parvovirus B19
a. Primarily infects RBCs
1) Entry through P antigen receptor
b. “Fifth disease” in children, red cell aplasia in adults
c. Nonenveloped, so not destroyed by solvent-detergent
treatment (concern in pooled treated products)
d. Source and recovered plasma for plasma derivative
manufacture are tested for Parvovirus via NAT
E. Other organisms for which we don’t test:
1. Prion disease
a. Prion: probably an infectious protein particle
b. Creutzfeldt-Jakob Disease (CJD)
1) Mostly sporadic (occasionally familial) spongiform
encephalopathy, nearly universally fatal
2) Found in older patients
3) Long disease course
4) Transmission via transfusion theoretical only
c. Variant CJD (vCJD)
1) Emerging syndrome in the United Kingdom
2) Caused by prion that causes bovine spongiform
encephalopathy (“mad cow” disease)
3) Distinct from CJD (younger, more rapid course)
4) Transfusion transmission proven
5) US deferral of many donors who lived in UK or
Europe since 1980 (see earlier)
6) Led to universal leukoreduction in many countries
(but current research suggests prion may also be
found in plasma)
2. Plasmodium species
a. Malaria is readily transmissible through transfusion.
b. No effective screening except by history.
c. See earlier for deferral guidelines
3. Babesia species
a. Tick-borne intraerythrocytic parasite infection
b. Huge concern in endemic areas in East currently; pilot
studies to test donors
c. Caused 10 cases of transfusion fatality from 2007-11
d. Screen via history (permanent deferral)

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APPENDIX I
Blood Donor Infectious Disease Screening Tests
Agent Screening Test(s) Confirmatory Test(s) Discussion
HIV  Anti-HIV 1/2  EIA/ChLIA: Western blot  RR anti-HIV + Reactive HIV NAT = permanent
(EIA/ChLIA) (WB) or IFA for HIV-1 deferral
 HIV-1 NAT (PCR,  EIA/ChLIA: HIV-2 EIA  RR anti-HIV + WB neg/indeterm + NR HIV NAT =
TMA) required after reactive indefinite deferral (may try to re-enter in 8 weeks)
anti-HIV-1/2  RR anti-HIV + positive WB = permanent deferral
 NAT: Individual donor  NR anti-HIV + reactive HIV NAT = indefinite deferral
NAT (if not done) (may try to re-enter in 8 weeks)
HCV  Anti-HCV  EIA/ChLIA: Repeat EIA  RR anti-HCV + reactive HCV NAT = permanent
(EIA/ChLIA) with another EIA (under deferral
 HCV NAT FDA variance) or  RR anti-HCV + RIBA neg/indeterm (unconfirmed
(PCR/TMA) approved supplemental supplement) + NR HCV NAT = indefinite deferral
NAT versions (may try to re-enter in 6 months)
 RIBA for anti-HCV EIA  RR anti-HCV + positive RIBA (confirmed
(not currently available) supplement) = permanent deferral
 NAT: Individual donor  RR anti-HCV + RR anti-HCV (different platform) OR
NAT (if not done) positive supplemental NAT = permanent deferral
 NR anti-HCV + reactive HCV NAT = indefinite
deferral (may try to re-enter in 6 months)
HBV  HBsAg  EIA/ChLIA:  RR anti-HBc x 1 = no deferral
(EIA/ChLIA) Neutralization for HBsAg,  RR anti-HBc x 2 = permanent deferral
 Anti-HBc none for anti-HBc  RR anti-HBc + RR HBsAg = permanent deferral
(EIA/ChLIA)  NAT: Individual donor  RR HBsAg + confirmed neutralization = permanent
 NAT HBV NAT (if not done) deferral
(Required 2013)  RR HBsAg + nonconfirmed neutralization = retest in >
8 weeks
 NAT HBV reactive + RR HBsAg (confirmed
neutralization) = permanent deferral
HTLV-I/II  Anti-HTLV-I/II  None licensed  Reactive anti-HTLV-I/II x 1 = no deferral
(EIA/ChLIA)  Reactive anti-HTLV-I/II x 2 = permanent deferral
Syphilis (T.  Many (hemag-  Usually FTA or TP-PA  Reactive screen + negative confirm = no definite
pallidum) glutination, EIA, deferral (though many will defer)
RPR)  Reactive screen + reactive confirm = at least 1 year
deferral (after treatment)
West Nile  WNV NAT  Individual donor NAT (if  Reactive NAT = 120 day deferral (if asymptomatic)
Virus (PCR/TMA) not done)
Chagas  T. cruzi  Enzyme Strip Assay  Reactive EIA = permanent deferral
Disease (T. EIA/ChLIA (ESA); FDA approved  ESA and RIPA results only for counseling
cruzi)  Many use RIPA (not FDA  No re-entry currently
approved)  Testing may be once per lifetime only
RR: Repeat reactive
EIA/ChLIA: Enzyme immunoassay or chemiluminescent immunoassay
PCR/TMA: Polymerase chain reaction or transcription-mediated amplification (available US NAT
platforms)
IFA: Immunofluoresence assay
RIBA: Recombinant immunoblot assay (NOTE: RIBA has been discontinued by manufacturer, and all
protocols using it are unavailable; see strikethrough text above)
FTA: Fluorescent treponemal antibody
TP-PA: Treponema pallidum particle agglutination
RIPA: Radioimmunoprecipitation assay
RPR: Rapid plasma reagin
Sources: AABB Technical Manual, 17th ed, www.fda.gov

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APPENDIX II
AABB Uniform Donor History Questionnaire (V 1.3, AABB 2008)
Question Comment
Are you:
1. Feeling healthy and well today?
2. Currently taking an antibiotic? Medical director discretion
3. Currently taking any other medication for an Same
infection?
Please read the Medication Deferral List
4. Are you now taking or have you ever taken any See deferrals listed
medications on the Medication Deferral List? previously in notes
5. Have you read the educational materials? Includes HIV risk info
In the past 48 hours
6. Have you taken aspirin or anything that has 48 hour deferral as sole
aspirin in it? source of platelets per FDA
In the past 6 weeks
7. Female donors: Have you been pregnant or are This question (as well as #24
you pregnant now? (Males: check “I am male.”) and #34) serves as a
“check” to make sure
donors are paying attention
In the past 8 weeks have you
8. Donated blood, platelets or plasma? See details in notes
9. Had any vaccinations or other shots? See notes
10. Had contact with someone who had a smallpox No deferral unless
vaccination? symptomatic. If so, defer at
least 14 days.
In the past 16 weeks
11. Have you donated a double unit of red cells 16 week donation interval
using an apheresis machine?
In the past 12 months have you
12. Had a blood transfusion? 1-year deferral
13. Had a transplant such as organ, tissue, or bone 1-year deferral
marrow?
14. Had a graft such as bone or skin? 1-year deferral (unless dura
mater, then permanent)
15. Come into contact with someone else’s blood? 1-year deferral
16. Had an accidental needle-stick? 1-year deferral
17. Had sexual contact with anyone who has 17-23 are all 1-year
HIV/AIDS or has had a positive test for the deferrals from the time of
HIV/AIDS virus? last contact
18. Had sexual contact with a prostitute or anyone
else who takes money or drugs or other payment
for sex?
19. Had sexual contact with anyone who has ever
used needles to take drugs or steroids, or
anything not prescribed by their doctor?
20. Had sexual contact with anyone who has
hemophilia or has used clotting factor
concentrates?

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21. Female donors: Had sexual contact with a male
who has ever had sexual contact with another
male? (Males: check “I am male.”)
22. Had sexual contact with a person who has
hepatitis?
23. Lived with a person who has hepatitis? 1 year deferral, unless
asymptomatic Hepatitis C
24. Had a tattoo? For 24-25, these can be
allowed if certified that
procedure was done by
state-certified entity using
sterile, one-time use needles
25. Had ear or body piercing?
26. Had or been treated for syphilis or gonorrhea? 1 year following treatment
completion (or from time of
positive test if no symptoms)
27. Been in juvenile detention, lockup, jail, or 72 hours is consecutive
prison for more than 72 hours?
In the past three years have you
28. Been outside the United States or Canada? Primarily for malaria
exposure or vCJD risk
From 1980 through 1996,
29. Did you spend time that adds up to three (3) For 29-32, see vCJD
months or more in the United Kingdom? discussion in notes;
(Review list of countries in the UK) permanent deferral
30. Were you a member of the U.S. military, a
civilian military employee, or a dependent of a
member of the U.S. military?
From 1980 to the present, did you
31. Spend time that adds up to five (5) years or
more in Europe? (Review list of countries in
Europe.)
32. Receive a blood transfusion in the United
Kingdom or France? (Review list of countries
in the UK.)
From 1977 to the present, have you
33. Received money, drugs, or other payment for Permanent deferral
sex?
34. Male donors: had sexual contact with another Currently controversial, but
male, even once? (Females: check “I am permanent deferral
female.”)
Have you EVER
35. Had a positive test for the HIV/AIDS virus? May require investigation to
determine if true positive. If
so, permanent deferral
36. Used needles to take drugs, steroids, or anything Permanent deferral.
not prescribed by your doctor? Includes obvious physical
stigmata of IVDA (needle
tracks)
37. Used clotting factor concentrates? Permanent if for hemophilia,
otherwise 1 year deferral
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38. Had hepatitis? Permanent deferral after
11th birthday
39. Had malaria? 3 year deferral
40. Had Chagas’ disease? Permanent deferral
41. Had babesiosis? Permanent deferral
42. Received a dura mater (or brain covering) graft? Permanent deferral for
vCJD possibility
43. Had any type of cancer, including leukemia? Medical director discretion
(see discussion in notes)
44. Had any problems with your heart or lungs? Medical director discretion
(see discussion in notes)
45. Had a bleeding condition or a blood disease? If hemophilia, permanent
deferral. Otherwise, medical
director discretion.
46. Had sexual contact with anyone who was born See HIV group O discussion
in or lived in Africa? in notes. Can be omitted if
anti-HIV test used detects
group O.
47. Been in Africa? HIV group O discusssion
48. Have any of your relatives had Creutzfeldt- Permanent deferral

Jakob disease?
Medication Deferral List

(Source: http://www.aabb.org/resources/donation/questionnaires/Pages/dhqaabb.aspx)
Please tell us if you are now taking or if you have EVER taken any of these medications:
 Proscar (finasteride)  usually given for prostate gland enlargement
 Avodart, Jalyn (dutasteride)  usually given for prostate enlargement
 Propecia (finasteride)  usually given for baldness
 Accutane, Absorica (Amnesteem, Claravis, Sotret, isotretinoin)  usually given for severe
acne
 Soriatane (acitretin) – usually given for severe psoriasis
 Tegison (etretinate) – usually given for severe psoriasis
 Growth Hormone from Human Pituitary Glands  used usually for children with delayed
or impaired growth
 Insulin from Cows (Bovine, or Beef, Insulin)  used to treat diabetes
 Hepatitis B Immune Globulin – given following an exposure to hepatitis B.
NOTE: This is different from the hepatitis B vaccine which is a series of 3 injections
given over a 6 month period to prevent future infection from exposures to hepatitis B.
 Plavix (clopidogrel) and Ticlid (ticlopidine) – inhibits platelet function; used to reduce the
chance for heart attack and stroke.
 Feldene – given for mild to moderate arthritis pain
 Experimental Medication or Unlicensed (Experimental) Vaccine – usually associated
with a research protocol

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Blood Bank III
D. Joe Chaffin, MD
Blood Components and Component Therapy

I. General
A. Basic concept of “component therapy”
1. More efficient use of products by giving a patient what he
needs and avoiding what he doesn’t need.
2. Made possible by advent of plastic bags around 1950.
3. Single unit may be made into numerous components (see
figure below representing classical US method).
B. Anticoagulant/preservative solutions
1. Allows blood to be stored for extended periods without
drastic effects on most metabolic and therapeutic qualities
2. Red cell storage defined by demonstrating 75% survival
of transfused cells at 24 hours after transfusion (FDA)
3. Historic anticoagulant/preservatives
a. Citrate-phosphate-dextrose (CPD) and citrate-
phosphate-dextrose-dextrose (CP2D)
1) Allow 21 days of RBC/whole blood storage
b. Citrate-phosphate-dextrose-adenine (CPDA-1)
1) Very similar to CPD but with 17.3 mg of adenine
(no adenine in CPD)
2) Allows 35 days of RBC/Whole Blood storage
c. Acid Citrate Dextrose (ACD): used for apheresis PLTs
4. Additive solutions (“Adenine Saline” additives)
a. Increases shelf life of RBCs to 42 days
b. Most common types
1) AS-1 (Adsol®)
2) AS-3 (Nutricel®)
3) AS-5 (Optisol®)
c. Specifics vary, but all add more dextrose and adenine
to increase blood shelf life.
d. AS-1 and AS-5 contain mannitol for RBC preservation
P}Chaffin (2/11/2013) Blood Bank III page 1
5. Preparation of additive solution RBCs:
a. RBCs with additive solution process:
1) Blood collected in CPD or CP2D (NOT CPDA-1),
spun, then mixed with 110 mL additive solution for
500 mL collections (100 mL for 450 mL bags)
2) This gives a product with more volume and less
plasma (HCT usually 55-65%)
6. Know storage details for various products (Table 1)
Table 1: Storage Details for Various Blood Products

Product Storage Product Storage
RBCs / 35 days (CPDA-1) Granu- 24 hours; 20-24 C
Whole blood 42 days (Additives) locytes (no agitation)
1-6 C
Fresh 1 year; –18 C OR
Frozen RBCs 10 years; –65 C; Frozen 7 years, –65 C;
24 hours after thaw Plasma 24 hours at 1-6 C
Washed 24 hours; 1-6 C after thaw
RBCs CRYO 1 year at –18 C
Platelets 5 days; 20-24 C 6 hours at 20-24 C
(gentle agitation); after thaw (4 hours
4 hours if pooled if pooled)
C. Quality control of blood products

1. Blood is a controlled product that is tightly regulated by
the FDA (with more regulations from AABB & CAP).
2. Very specific, detailed requirements acceptability
Table 2: Quality Control for Blood Products (US)

Product QC Product QC
RBCs HCT < 80% (all), > 50 g Apheresis ≥ 3.0 x 1011 and pH ≥ 6.2 in
HGB in 95% (apheresis platelets 90%
RBCs)
RBCs ≤ 5 x 106 WBCs in 95%, Apheresis Above + < 5.0 x 106
leukoreduced retain 85% of RBCs platelets residual WBCs in 95%
leukoreduced
Platelets (PC) ≥ 5.5 x 1010 and pH ≥ 6.2 CRYO Factor VIII ≥ 80 IU (all)
in 90% Fibrinogen ≥ 150 mg (all)
Platelets (PC) ≥ 5.5 x 1010 in 75%, pH ≥ Granulocyte ≥ 1.0 x 1010 in 75%
leukoreduced 6.2 in 90%, AND < 8.3 x concentrate
105 WBCs in 95%
II. Blood and Components

A. Blood components
1. Red blood cells/additive solution red blood cells
2. Platelets
a. Whole blood-derived platelets (WBD-PLTs)
b. Apheresis-derived platelets (AD-PLTs)
3. Modified RBCs and platelets
a. Leukocyte reduced products
page 2 Blood Bank III P}Chaffin (2/11/2013)

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b. Irradiated products
c. Frozen products
d. Washed products
4. Plasma and derivatives
a. Fresh frozen plasma (FFP)
b. FFP alternatives (including FP24)
c. Cryoprecipitate (“antihemophilic factor”)
d. Factor concentrates
e. Other plasma derivatives
5. Miscellaneous products
a. Granulocyte concentrate
b. DDAVP
c. Recombinant activated factor VII (NovoSeven)
B. Whole blood
1. The original blood product!
2. Minimal availability in most blood banks today
3. Specifics:
4. Potential indications:
a. Massive blood loss (30-40% or more of blood volume)
1) Trauma/emergency transfusions most commonly
2) Use may lead to less exposure by providing coag
factors (and maybe a few functional platelets), as
well as volume
3) Whole blood must be ABO identical due to
plasma; tougher to use in emergencies.
b. Exchange transfusions in neonates (more often
“reconstituted” from separate RBCs and FFP)
c. Autologous transfusions
5. Contraindications:
a. Anything where something more specific to the
patient’s needs would be better.
6. Storage Time and Conditions
a. Length depends on anticoagulant/preservative used
b. 1-6oC.
C. Red blood cells (with and without additives)
1. The most commonly used blood component
2. Prepared by centrifugation and removal of most of plasma
layer of whole blood, or by apheresis collection.
a. May be transfused without modification after
preparation or may use additive solution

3. Specifics:
4. Requirements:
a. HCT < 80% for all RBCs (easy with AS-RBCs)
b. Apheresis RBCs: 95% must have >50 g HGB or 150
mL of RBCs
5. Indications
a. Need for increased oxygen-carrying capacity
1) Deciding if RBC transfusion is indicated
a) Balance risks of anemia vs. risks of transfusion
b) Hemoglobin level alone is a very inaccurate
indicator of the need for transfusion
c) Anemia compensation (HGB dissociation curve
shift to right, inc. cardiac output, dec. blood
viscosity, inc. respirations, etc.) is robust
d) Cardiac factors, O2 demand often overlooked
e) Measuring mixed venous saturation (SvO2) and
comparing to arterial levels (SaO2) gives an
estimate of current oxygen use
• Example: 25% extraction (SaO2 100%, SvO2
75%) is normal; extraction may go up to 75%
or more when necessary (exercise, etc)
• Heart muscle has little reserve; extracts close
to 75% normally
• Overall oxygen extraction ratio of 0.5 (50%)
or more at rest is deemed “critical.”
f) All factors (including blood volume, heart
function, ability to increase cardiac output, and
O2 requirements) should be addressed when
considering transfusion.
• Due to compensation (including increased
blood volume), chronic anemia is less likely
to need transfusion (and may be dangerous!)
2) Situations that may require red cell transfusion:
a) Acute hemorrhage (over 30% of blood volume
acutely)
b) Hemolysis
c) Marrow failure
b. Exchange transfusions
1) Sickle cell patients (esp. crisis or presurgery)
2) Hemolytic disease of the newborn/fetus (HDFN)

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c. Thresholds
1) Using a universal threshold like HGB 10 g/dL, HCT
30% is outdated, silly, and bad practice
2) All specialties have published recommendations
that agree with statement above
3) General guidelines:
• If HGB < 6 g/dL, transfusion usually needed
• If HGB >10 g/dL, transfusion rarely needed
• If HGB is between 6 and 10, clinical judgment,
assessment of situation, etc, is required
4) Based on published studies:
• It is reasonable to withhold transfusion in
orthopedic or ICU patients (non-acute MI) until
HGB is below 7-8 g/dL
• It is reasonable to transfuse to higher threshold
after acute MI (9-10 g/dL HGB)
• It is reasonable to assess need for further
transfusion after each unit given
6. Contraindications
a. Acute hemorrhage < 20% of blood volume
1) Crystalloids are adequate in most of these cases
b. Chronic nutritional anemias (folate, B12, iron)
7. Expected effect (per unit)
a. HCT increases 3%, HGB 1 g/dL (without acute
bleeding or hemolysis)
b. Effect can be measured 15 minutes after transfusion
8. ABO compatibility
a. ABO type of transfused RBCs must be compatible
with recipient plasma ABO antibodies
b. Always protect the transfused cells! (See chart)
9. Storage and shipping

a. Same as for whole blood if CPD, CPDA-1 used
b. 42 days at 1-6 C if additive solutions used
c. Shipping temperature 1-10 C
10. Compatible fluids
a. Normal saline (0.9%, not 0.45%)
b. ABO compatible plasma
c. 5% albumin
d. Normosol-R pH 7.4 and Plasma-Lyte 148 and -A
d. Red cells should not contact lactated Ringer’s (LR),
D5W, 0.45% NS, antibiotics/other drugs, or TPN
1) Hypotonic solutions - red cells swell and burst;
hypertonic solutions - red cell shrinkage.
2) LR has enough calcium to counteract the citrate
anticoagulant in blood (LR has 3 mEq Ca2+/L)
11. Red cell types (most covered in other parts of handout)
a. Red Blood Cells, Low Volume
1) Prepared from whole blood collection 66-90% of
the target volume (for 500 mL bags, 333-449 mL)
2) RBCs may still be used, but not plasma
b. Red Blood Cells, Adenine Saline Added
c. Red Blood Cells, Leukocyte-reduced
d. Red Blood Cells, Frozen
e. Red Blood Cells, Deglycerolized, and Red Blood
Cells, Washed
f. Red Blood Cells, Irradiated
g. Red Blood Cells, Apheresis (often “double” products)
h. Red Blood Cells, Rejuvenated (not currently available)
1) RBCs may be “rejuvenated” up to 3 days after
expiration in CPD or CPDA-1, or up to the
expiration date in AS-1 with “Rejuvesol.”
2) Restores ATP, 2,3-DPG levels to near-normal levels
3) Rejuvenated product is often frozen (up to 10 years
for CPD, CPDA-1 RBCs; up to 3 years for AS-1)
4) Thawed (or immediately transfused) product must
be washed, then transfused within 24 hours.
D. Platelets (See Oct 2010 podcast)
1. Whole blood-derived platelets (platelet concentrate,
“random platelets”, WBD-PLTs)
a. Prepared via centrifugation (“soft” spin then “hard”
spin in the US) from a single whole blood unit.
b. May be pooled at blood center or transfusion service

c. Specifics
d. Traditional dose
1) 1 unit per 10 Kg body weight
a) Typically given six bags at a time in adults

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2) 10-15 mL/Kg in neonates
2. Apheresis platelets (“single donor”, “AD-PLTs”)
a. Made from one donor via apheresis procedure
1) Apheresis = removing whole blood from body,
taking what you want, then returning the rest.
2) Roughly 85% of platelets transfused in the US
3) Double or triple products can come from 1 donor
(each must have > 3.0 x 1011 PLTs)
b. Specifics:
c. Why choose AD-PLTs over WBD-PLTs?

1) Limiting exposure
a) For infectious disease transmission
• 1 exposure vs. at least six for PC decreases
risk of viral transmission
• Also decreases risk of bacterial contamination
b) For HLA immunization?
• Fewer donor exposures = less immunization?
• No! Risk more dependent on exposure to
donor WBCs (not number of donors)
• “TRAP” study: NEJM. 1997;337, No. 26,
1861-9.
2) Platelet refractoriness (see BB Practical)
a) Lack of response to platelet transfusion (immune
and nonimmune causes)
b) May be used as HLA-matched or crossmatched
doses for immune refractoriness.
3. Indications for platelet transfusion:
a. Thrombocytopenia
1) Transfusions may be prophylactic or therapeutic
2) Prophylactic
a) Data supports a prophylactic threshold of <5,000
(but most use 10K).
b) 20K threshold if patient has risk factors
i) Fever, sepsis, bleeding, thrombocytopathy
c) 50K if about to have major surgery
3) Therapeutic
a) 50K if bleeding
b) 100K reasonable for patients with intracranial
and pulmonary hemorrhage
4) Controversy in LP, liver biopsy, endoscopy
a) Many use 50K or 100K prophylactic threshold
b) Not proven; count not predictive of bleeding
c) Outcome is operator-skill dependent!

b. Thrombocytopathy
1) Prophylactic transfusions not indicated
2) Therapeutic indications:
a) Congenital defects with bleeding
b) Drugs (Plavix, ASA most common)
• Increased bleeding in emergency surgical
patients, especially cardiac surgery
c) External agents
• Cardiac bypass
• ECMO
d) Metabolic effects (e.g., chronic renal failure)
• PLTs are not first-line defense! (dialysis,
DDAVP, Cryo, conjugated estrogens, etc).
4. Contraindications to platelet transfusion
a. Thrombotic thrombocytopenic purpura (TTP)
1) ADAMTS13 enzyme deficiency most common
leading to large vWF multimers and subsequent
platelet microthrombi
2) More platelets could lead to more thrombi
3) May be overblown fear; many use PLTs in life-
threatening situations without problems.
4) Hemolytic-uremic syndrome (HUS) similar to TTP
without neurologic symptoms
b. Heparin-induced thrombocytopenia, type II
1) Antibody vs. heparin/platelet factor 4 complex
2) These patients are at great risk for thrombosis, and
platelets should be avoided if possible
c. Immune/idiopathic thrombocytopenic purpura
(relative)
1) Doesn’t help count, and patients don’t usually bleed
2) Use only if significant bleeding occurs
d. Post-transfusion Purpura (PTP); see BB IV
1) Uncommon antibody vs. transfused PLTs
2) Most transfused PLTs will be antigen-positive
5. General comments about platelets
a. Dose
1) WBD-PLTs 1 bag/10 Kg weight (4-6 bags/dose
traditional)
2) AD-PLTs 1 bag/dose (assuming 3.0 x 1011 count)
3) Recent study: No outcome diff. with < 3.0 x 1011
4) Neonates: 10-15 mL/Kg (may be concentrated)
b. Expected effect
1) Generally, if one hour post-count increases by
>20,000-30,000, response is adequate (“eyeball”)
2) CCI has value in determining adequacy of response,
even if count doesn’t increase much (see BB Prac.)
3) One-hour post-transfusion count is standard.
c. Storage and shipping
1) 5 days at 20-24 C (with gentle agitation)

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2) Shipping range “as close as possible” to 20-24 C
3) < 24 hours without agitation during shipping
4) 4 hours after transfusion service pooling (open
system; “pre-pooled” PLTS have normal shelf life)
d. ABO and RhD
1) PLTs do not require pretransfusion crossmatches,
and ABO-incompatible platelets commonly given.
2) RhD antigens are NOT present on platelets.
a) Contaminating RBCs have D antigen (esp WBD
PLTs)
b) Risk of making anti-D is low without
prophylaxis (3.8% in recent ten-year study)
b) Consider Rh prophylaxis in premenopausal D-
neg women (1 vial RhIG per 2-3 weeks;
effective as long as anti-D is detectable)
3) Platelet ABO incompatibilities
a) Major = platelet ABO antigens incompatible
with recipient plasma (like A plts to O recip)
• Cleared from circulation faster; less effect
b) Minor = donor ABO antibodies incompatible
with recipient RBCs (like O plts to A recipient)
• A concern in children, neonates, and high-
titer donors (“reverse” hemolytic reactions)
c) ABO-identical is best, but not always practical
e. Platelet sterility
1) AABB Standards requires centers to both limit and
detect bacterial contamination of ALL platelets
2) Limiting contamination
a) Careful skin preparation
b) Discarding initial 20-30 cc of blood (done
automatically with diversion pouches)
c) Exclusive use of apheresis platelets
3) Detecting contamination
a) Culture-based methods
• Require 24 hour wait before taking sample
• BacT/ALERT (bioMerieux, Inc)
• Enhanced Bacterial Detection System (eBDS)
b) Pre-issue bacterial detection
• Verax PGD (Verax Biomedical); approved
for leukoreduced AD-Platelets and pooled
WBD-platelets
• BacTx (immunetics); approved for
transfusion service-pooled WBD-platelets
c) Old (no longer accepted) methods
• Gram stain, swirling, glucose checks
E. Modifications to red cells and platelets
1. Leukocyte reduction (LR)
a. Definitions (as of AABB Standards, 26th ed.)
1) In US: ≤ 5 x 106 residual WBCs

2) In Europe: ≤ 1 x 106 residual WBCs
3) US rules
a) ≤ 5 x 106 white cells in 95% of tested units
defines leukocyte-reduced:
• RBCs, AD-PLTs, and whole blood
b) ≤ 8.3 x 105 WBCs in 95% of tested units defines
leukocyte-reduced:
• WBD-PLTs (NOTE: 8.3 x 105 x 6 = 5 x 106)
c) Each must also retain at least 85% of original
component and meet all other QC standards.
b. Methods
1) Leukocyte reduction filters
a) 99.99% of white cells (“4 log” reduction)
2) Apheresis collection devices
a) Built-in leukoreduction methods.
b) Methods vary by manufacturer.
c. “Universal” LR is not mandated in the US, but vast
majority of cellular components are leukoreduced
d. Types
1) “Prestorage” leukocyte reduction
a) Usually < 72 hours after draw (always <5 days).
b) Inline filters at time of collection or post
collection filters for red cells.
c) Apheresis LR is prestorage by definition
2) “Pretransfusion” leukocyte reduction
a) Immediately prior to transfusion (“bedside”)
• Least desirable
• Lack of available QC, poor training
• Many older studies done with this method
b) Better done in transfusion service before issuing
e. Established benefits:
1) Prevention of febrile nonhemolytic transfusion
reactions
a) Benign reactions, but mimic early hemolysis
b) First type: WBCs secrete pyrogenic cytokines in
bag before transfusion: Common with PLTs
c) Second type: Pyrogenic cytokines secreted after
transfusion.
• Seen more commonly with RBC transfusions
• Recipient antibodies against transfused WBC
antigens or immune complexes of donor
WBCs and coating antibodies binding to
macrophages
d) Prestorage LR prevents both types,
pretransfusion only the second type
2) Prevention of HLA immunization
a) HLA antibody formation requires HLA class I
antigens be presented by transfused lymphocytes
b) LR works well to prevent this interaction

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c) Use for multiple transfusion recipients
d) UV-B treatment also works; not available in US.
3) Prevention of CMV transmission
a) Virus carried only in a small minority of donor
WBCs (monocytes).
b) Blood. 1995;86,3598-3603; “The Bowden
Study” – landmark LR study
• Filtered products equivalent to CMV
seronegative in preventing seroconversion.
• Neither method is perfect; very early infection
may escape testing and/or leukoreduction
c) See BB Practical for further discussion
f. Potential benefits:
1) Reduction of reperfusion injury post cardiac
bypass (well-proven now)
2) Prevention of immunosuppressive effects of
transfusion
a) Controversial, not universally accepted
b) Transfusion seems to immunosuppress recipient
(“transfusion-related immunomodulation” or
TRIM).
• Increased post-op infections,cancer
recurrence, mortality in transfused patients
c) Donor WBCs may be the cause, but studies since
LR widespread has not proven effect
3) Reduction of bacterial/parasitic contamination
a) Some studies suggest reduction in organisms
like Yersinia or Leishmania with leukoreduction.
b) Probably not reliable enough to depend on!
4) Reduction in the risk of prion disease
a) One of the reasons for universal LR in Europe
b) Current: Prion known to be in plasma, too
c) Prion-specific filters developed
g. Contraindications:
1) Prevention of transfusion-associated GVHD.
a) Irradiation is only proven method (cases of TA-
GVHD with leukoreduced blood reported)
2) Transfusion of granulocyte concentrate
3) Transfusion of previously frozen products (FFP,
cryo, etc); most consider unnecessary
2. Washing
a. 1-2 L of saline removes about 99% of plasma.
b. Generally takes one to several hours (automated)
c. Shelf life
1) Red cells: 24 hours post-wash
2) Platelets: 4 hours post-wash
d. Why bother?
1) Removal of plasma proteins for hypersensitivity
(RBCs and platelets)

a) Classic example: IgA deficiency
• A few IgA deficient patients develop anti-
IgA; exposure leads to anaphylaxis.
• Requires intense washing (3L or so)
• IgA-deficient donors are alternative
b) Removal of unwanted antibodies
• ABO antibodies (neonatal transfusions)
• T-activation (polyagglutination)
2) Neonatal alloimmune thrombocytopenia (NAT,
NAIT, FNAIT)
a) Severe thrombocytopenia usually due to
maternal anti-HPA-1A (80%); similar to HDFN
in concept
• Exposure to antigen via pregnancy or
transfusion, antibody formed
• IgG crosses placenta, attacks baby’s platelets
• 25% occur in 1st pregnancy (rapid antibody
formation)
• 10-30% get intracranial hemorrhage
b) Maternal IVIG used before birth (+/- intrauterine
platelet transfusion)
c) Platelet choices:
• HPA-1A negative platelets given after birth
• Washed, irradiated maternal platelets may
also be used (lack antigen and antibody)
3) Removal of unwanted electrolytes (RBCs/PLTs)
a) Especially in neonatal transfusions
b) Large-volume or irradiated products
3. Freezing
a. Cryopreservative agents protect component while
freezing and thawing
1) Most common: Glycerol at 40% concentration
2) Deglycerolization before transfusion = washing
3) DMSO for platelets, but recovery is very poor (1/3)
b. Why bother?
1) Storage of rare, autologous, or O-negative units
2) Plasma hypersensitivities (as with washed)
3) Repeated febrile reactions (as with washed)
c. Storage
1) Before freezing:
a) CPD, CPDA-1, or CP2D: Maximum 6 day shelf
life before glycerolization
b) Additive solutions (AS-1, AS-3, AS-5): Up to
full 42 day shelf life before glycerolization
2) Red Cells
a) 10 years at –65 C (40% glycerol)
b) 24 hours at 1-6 C after thawing/deglycerolizing
3) Platelets
a) At least two years at -80 C (not FDA licensed)

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4. Irradiation of cellular components (more in BB4)
a. Irradiation dose deactivates T-lymphocytes
b. Prevent transfusion-associated graft vs. host disease.
c. Radiation dose
1) 2500 cGy (“rad”) dose required targeted to center of
bag, with at least 1500 cGy in all parts of the bag
d. Indications for irradiation (details in BB4)
1) Immunosuppression
a) Congenital T-cell deficiencies (DiGeorge’s,
SCID, Wiskott-Aldrich)
b) Stem cell or marrow transplant recipients
c) Patients taking chemo agents that attack T-cells
(Fludarabine, other purine analogs)
d) Aplastic anemia patients
e) Patients with solid tumors getting intensive
chemotherapy/radiation
2) Intrauterine transfusions, premature neonatal
transfusions, and neonatal exchange transfusions
3) Hematologic malignancies (esp. Hodgkin’s)
4) Granulocyte transfusions
5) Receiving blood from a first-degree relative
donor or receiving HLA-matched units
e. Patients probably NOT at risk (but often get irradiated
products anyway).
1) Solid organ transplant recipients
2) Term neonates
3) AIDS patients
4) Patients receiving previously frozen blood products
(FFP, cryoprecipitate)
f. Maximum storage: 28 days after irradiation or regular
expiration date, whichever comes first
1) K+ triples and free hemoglobin increases in plasma,
indicative of mild RBC membrane damage
F. Plasma Group (See Mar 2010 Podcast)
1. Fresh frozen plasma (FFP)
a. Widely used but not well-studied prospectively
b. Whole blood-derived (see earlier diagram) or from
apheresis (AD-FFP can have much higher volume)
c. Specifics for WBD-FFP:
d. General notes about coagulation and FFP transfusion

1) 30-40% of coag factors required for hemostasis.

2) “Adequate” factor levels don’t necessarily give
normal coag tests (mild elevations common)
3) Most FFP transfusions are given for elevated
PT/INR levels (Factor VII most responsible)
a) Factor VII in-vivo half-life is only about 4 hours
b) The INR of FFP is roughly 1.5!
4) Prophylactic FFP use with mild lab elevations is
usually a mistake, even before procedures
a) No evidence of bleeding prevention or lab value
correction when these patients are transfused
5) No universal threshold exists, but many use INR of
2.0 as indicator of serious factor deficiency
e. Indications
1) Bleeding patients with coagulopathy due to
multiple factor deficiencies
a) Hepatic failure
• Decreased production of all hepatic factors
(including pro- and anticoagulants)
• PT doesn’t predict bleeding; avoid
prophylaxis
b) Dilution from massive transfusion
c) Consumptive processes (DIC)
2) Bleeding patients requiring urgent reversal of
vitamin K deficiency from warfarin effect
a) Warfarin affects factors II, VII, IX, X
b) IV or SQ vitamin K takes hours (between 6 and
12) to replenish these factors (oral.
c) For non-bleeding patients, correct without FFP
administration (hold dose and give vitamin K)
d) In bleeding patients, Prothrombin Complex
Concentrate (PCC) may be better choice than
FFP (but will still need FFP since US PCC preps
have minimal FVII)
e) In general, need at least 10-20 ml/Kg of FFP to
attain hemostasis in these patients (usually more)
f) Chest 2008;133:160S-198S; get this article!
3) Trauma transfusion
a) Recent literature suggesting that trauma patients
given plasma in close to 1:1 ratio with RBCs
have better survival (first reported in military)
b) Much current argument pro and con
c) Many trauma centers have established
“trauma/massive transfusion protocols” that
attempt to make 1:1 ratio automatic
d) Concerns about plasma transfusion
complications (TRALI) and AB plasma wastage
e) Not proven in randomized, prospective studies

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4) Dilutional coagulopathy
a) Transfusion of multiple coag factor poor
products (RBCs and crystalloids in massive
transfusion) dilutes coag factors.
b) Usually not apparent until after at least 10-15 or
more units of RBCs (with accompanying fluid)
in a 24 hour time span
c) May be less of an issue with massive transfusion
protocols and 1:1 ratios mentioned above
5) Transfusion or plasma exchange for TTP/HUS
a) Acquired or congenital ADAMTS13 deficiency;
large vWF multimers lead to platelet thrombi
b) FFP has normal amounts of ADAMTS13
c) Plasma exchange standard treatment
d) Treatment until PLT count is at least 100K with
near-normal LDH
6) Other factor-specific coagulopathies without an
available factor concentrate (V, X, XI in US)
7) C1-esterase inhibitor deficiency
f. Contraindications:
1) Volume expansion
a) Albumin, crystalloids are safer.
2) Heparin reversal
a) Antithrombin, which potentiates heparin!
b) Use protamine sulfate or just stop the heparin.
3) Factor deficiencies with available concentrates
4) Prophylactic or pre-procedure treatment of mild
elevations of PT/PTT; see above
5) “Nutrition,” “Wound healing,” or “well-being”
g. Preparation/storage
1) Pre-storage
a) Separated and placed at -18 C within 8 hours
b) NOTE: FFP does not have to be completely
frozen at 8 hours, just in -18C environment
2) Pre-transfusion
a) Stored at -18C up to 1 year (or -65C for 7 yrs)
b) Thawed at 30-37C (water bath/approved device)
c) Stored after thawing at 1-6 C for 24 hours
• FDA historically allowed 6 hours; now allows
24 hrs as specified in Circular of Info
• May extend beyond 24 hours per AABB (see
“thawed plasma” below)
g. Dosage
1) Commonly given two bags at a time in adults
2) 10-20 mL/Kg more appropriate (3-7 bags if 70 Kg)
3) 10-15 mL/Kg appropriate dose in neonates.
h. Effect
1) Standard dose increases factor levels by about 20-
30% in a 70 Kg person.

2) Transient due to short half-lives (FVII)
3) Greatly elevated PT/PTT more affected than mild
4) Will not help if INR is <1.5-1.6!
i. ABO and Rh
1) Donor antibodies compatible with recipient RBCs.
2) Give without regard to Rh.
2. Plasma variants
a. Plasma frozen within 24 hours of phlebotomy
(FP24 or PF24)
1) Not frozen in 8 hours like FFP, but 24 hours
2) Factor V levels essentially equal to those in FFP,
while factor VIII levels decline 20-25% vs. FFP
3) Stored and managed just like FFP (including
“thawed plasma” conversion below)
4) Except for DIC patients, can be used identically to
FFP (low FV and/or FVIII is uncommon)
b. “Thawed Plasma”
1) FFP/FP24, once thawed, is only good for 24 hours
2) Thawed FFP/FP24 may be relabeled as “Thawed
Plasma” and kept at 1-6 C for up to 5 days
a) Process is outlined in Circular of Information
but is not recognized by FDA
3) Indications are essentially identical to FFP, despite
a decrease in FV and FVIII to ~50% by 5 days
c. Plasma, cryoprecipitate reduced (“cryo-reduced
plasma”, “cryosupernatant”)
1) Residual plasma that remains after cryoprecipitate
harvested from FFP (see below).
2) Decreased levels of stuff that is in CRYO (FVIII,
fibrinogen, vWF, FXIII)
3) Sole indication: TTP patients (due to less vWF),
used in plasma exchanges if regular FFP doesn’t
work (literature shows mixed results on this).
4) Storage and transfusion just like FFP.
d. Source plasma
1) Apheresis collection, usually paid donors
2) Used for manufacture, not transfusion
3) Licensed product
e. Recovered plasma
1) Plasma from volunteer whole blood donation

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2) Unused units of frozen plasma may be relabeled as
recovered and sold for further manufacture
(albumin, etc) under short supply agreement
3) Unlicensed product, source of revenue for blood
centers
f. Liquid plasma/plasma
1) Plasma separated from whole blood up to 5 days
after expiration
2) Liquid plasma: stored at 1-6 C, not frozen
a) Can be transfused up to 5 days after whole blood
expiration date, but rarely used
3) Plasma: stored at -18 C or below; rarely used
3. Cryoprecipitate
a. Also has seen increased use in recent years.
b. Specifics:
c. QC Requirements:
1) > 80 IU FVIII per bag
2) > 150 mg fibrinogen per bag (easy! Most contain at
least 250 mg)
d. Indications
1) Fibrinogen deficiency (congenital or acquired)
a) General threshold: 100 mg/dl for adequate
hemostasis post-surgery.
b) Calculation in BB Practical section
c) Many use 10-20 bags per dose in adults, more if
fibrinogen is less than 50 mg/dl.
d) 10 bags deliver about 2500 mg of fibrinogen in
about 150 ml of volume
• > 1 liter FFP needed for same amount!
2) Treatment of uremic thrombocytopathy
a) Acquired adhesion defect (probably) which may
respond to vWF supplementation
b) Generally seen with creatinine levels > 3 mg/dL
c) Second line of defense (after DDAVP, dialysis)
d) Also: Conjug. estrogens, inc. HCT to ~30%
e) Am J Med. 1994;96:168-79 describes treatment
of uremic thrombocytopathy.
3) Factor XIII deficiency (if concentrate unavailable)
4) Topical “glue”
a) Historically mixed with bovine thrombin and
applied directly to raw surfaces
b) Currently available fibrin sealants (treated,
virus-free) have made this less common.
5) Treatment of von Willebrand’s disease
a) Second-line therapy; should be used only if
factor VIII concentrates are not available.
• Some factor VIII concentrates (e.g., “Humate-
P”) contain vWF.
b) Cryo may be used for severe forms.
• Dose 1 bag per 10 Kg body weight q 8 hr
c) DDAVP can be used for milder forms.
6) Treatment of hemophilia A
a) Use only if emergency and no factor VIII
concentrate available.
b) Calculation in BB Practical section for exams
e. Manufacture
1) Made from a single unit of FFP.
2) Thaw FFP at 1-6 C, spin and remove liquid, re-
freeze slushy precipitate within 24 hours.
3) Commonly “pre-pooled” (before storage) under
sterile conditions at blood centers (variants: 4, 5, 8,
10 bags most common)
f. Storage and preparation for transfusion
1) -18 C for 1 year
2) After thawing (at 30-37 C, like FFP), store up to 6
hours at 20-24 C (unlike FFP)
a) Pre-pooled cryo units mentioned above have a 6
hour shelf life after thawing
3) If units are pooled without sterile docking
equipment, transfuse within 4 hours.
4) No compatibility testing required
5) ABO-compatible is preferred by some, but paucity
of anti-A/B makes it really not important
6) Can give without regard to Rh status
g. Myths
1) Cryo is NOT just small volume FFP
a) Common misconception
b) Can’t replace FFP in volume sensitive patients
2) There is NOT more fibrinogen in Cryo than in FFP
a) What’s there is more concentrated
4. Factor concentrates (a few)
a. Factor VIII concentrate
1) Used for moderate to severe hemophilia A
2) Virus inactivated or recombinant
3) Dosage: discussed in BB Practical
4) Target levels: as above
5) May contain vWF and be used in vWD.
b. Factor IX concentrate
1) Used for hemophilia B
2) Virus inactivated or recombinant
3) NOT the same as Factor IX Complex Concentrate

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c. Prothrombin Complex Concentrate (PCC)
1) Or, “Factor IX Complex Concentrate”
2) Approved only for bleeding hemophilia B patients,
but USED in warfarin overdose correction
3) Not “activated” as in the past; much less thrombosis
4) U.S. versions lack Factor VII, which limits utility
5. Albumin and plasma protein fraction
a. Volume expanders
b. Virus inactivated
c. Ridiculously expensive!
d. Differ only in composition
1) Albumin: 96% albumin, 4% globulins/others
2) PPF: 83% albumin, 17% globulins/others.
G. Granulocyte concentrate
1. Increasing use due to use of donor stimulation
2. Effect not definitively proven (current large study)
3. Specifics:
3. Indications
a. Consider in premature neonates with sepsis or
infections, transplant patients with infections, patients
with chronic granulomatous disease
b. Aside from above, a clinical situation including:
1) Fever for 24-48 hours,
2) Proven bacterial or fungal infection
3) No response to antibiotic therapy
4) Neutropenia (<500/uL; <3000/uL in neonates)
5) Reversible bone marrow hypoplasia
5. Not currently indicated for:
a. Prophylactic use
b. Patients with no hope of marrow recovery
6. 1.0 x 1010 is minimum yield (required in 75%), but many
centers are stimulating volunteer donors with G-CSF (+/-
steroids) for much greater yield; this is not FDA-approved
7. Cans and Can’ts!
a. Can (and should) irradiate to prevent TA-GVHD.
1) Irradiation deactivates T-lymphs but not PMNs.
b. Can’t filter to prevent CMV transmission.
1) This seems obvious, doesn’t it?
2) Use CMV-negative donors for “CMV-safe”
8. Storage conditions
a. 24 hours from collection at 20-24 C, without agitation
9. Cautions
a. Must be ABO, Rh, and crossmatch compatible

b. Most are transfused before inf. disease testing is done;
recently tested apheresis PLT donors commonly used
H. DDAVP (Desmopressin)
1. Synthetic ADH used for treatment of diabetes insipidus.
2. Causes release of vWF from endothelial cells;
functionally increases factor VIII, as well.
3. Potential indications:
a. Uremic thrombocytopathy
1) 0.3-0.4 g/Kg IV x 1; repeat with caution
2) Should be considered before platelets or CRYO.
b. Mild hemophilia A (>5% FVIII levels) and von
Willebrand’s disease (type I)
1) 0.3 g/Kg IV x over 30 min (30 min before
surgery); repeat with caution
4. Effect decreases with doses >q 48 hrs (“tachyphylaxis”)
I. Recombinant activated factor VII (NovoSeven)
1. Non-human-plasma-derived product that is currently
FDA-approved for use in:
a. Hemophiliacs (A or B) with inhibitors (bleeding
prevention and bleeding treatment)
b. Patients with congenital factor VII deficiency
(bleeding prevention and bleeding treatment)
2. Widespread “off-label” use has occurred, as NovoSeven
gained traction as a “magical” hemostatic agent!
3. Estimated 1-2% risk of thrombosis is concerning
a. JAMA. 2006;295:293-298 (O’Connell, et al) article
reported that most serious thromboembolic
complications from NovoSeven followed off-label use.
b. Thrombotic stroke, acute MI, and pulmonary emboli;
NOTE that these were not definitely caused by
NovoSeven, just associated with its use.
4. Off-label use may be most appropriate for:
a. Treat/prevent surgical bleeding in trauma patients
b. Reversal of anticoagulant therapy (warfarin and factor
Xa inhibitors)
c. Treat/prevent surgical bleeding in advanced liver
failure patients.
d. Perioperative blood loss prevention (after failed
clotting factor replacement therapy) in cardiac surgery,
neurosurgery, OB/GYN surgery, and urologic surgery
5. Many other off-label uses are being studied.
6. Not indicated for routine pre-procedure prophylaxis.
7. Typical doses: 20 to 40 mcg/Kg in non-emergencies, 41
to 90 mcg/Kg otherwise
a. Roughly 2-hour half-life, repeat dose often

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Blood Bank IV
D. Joe Chaffin, MD
Transfusion Reactions
A. Scope of the problem
1. Transfusions still harm, despite great reductions in
transfusion-transmitted diseases
Figure 1: FDA Transfusion Fatalities FY 2007-2011

(Source: http://www.fda.gov)
B. Suspected reaction workup

1. When?
a. Indicated when possible reaction is suspected by a
combination of signs/symptoms
b. Just a few:
1) Inflammatory:
a) Fever/chills, skin changes, infusion site pain
2) Circulatory:
a) BP changes, shock, hemoglobinemia/uria
3) Pulmonary:
a) Dyspnea, orthopnea, wheezing, full failure
4) Coagulation:
a) Unexplained increase in bleeding, DIC
5) Psychological:
a) Sense of unease or impending “doom”!
2. General philosophy (my opinions):
a. Opinion 1: Assume all suspected reactions are
hemolytic, and work to disprove your assumption
b. Opinion 2: Anyone involved in a transfusion should be
allowed to initiate a transfusion reaction workup
3. STEP ONE: STOP THE TRANSFUSION!
a. Don’t disconnect the unit (though that will eventually
happen); at least stop the incoming flow of blood.
b. Main indicator of survival of an acute HTR:
amount of incompatible blood infused
P}Chaffin (2/11/2013) Blood Bank IV page 1

c. Leave the line open with saline.
Figure 2: Transfusion Reaction Workup

4. Necessary parts of workup (things everyone should do):
a. Clerical check
1) Bedside paperwork/bag check
2) Blood bank paperwork/computer check
3) Includes basic inspection of the unit for
discoloration or obvious issues
a) Darkened color: Susp. of bacterial contamination
b) Clots, aggregates, or anything out of the ordinary
b. Visible hemoglobinemia check
1) Spin a post-transfusion EDTA sample and examine
visually for a pink-red color change
2) Compare to pretransfusion sample if abnormal.
3) Detects > 2.5 to 5 ml of hemolysis occurring
anywhere in the body
4) Most sensitive way to detect intravascular
hemolysis; not specific, though
c. Direct antiglobulin (Coombs) test (DAT)
1) Demonstrates coating of RBCs with antibody and/or
complement in-vivo (see figure 3 below)
2) Most commonly polyspecific method (IgG + C3d)
3) If positive, compare to pretransfusion DAT
4) Positive DAT does not prove an acute HTR
a) Nonspecific positives in hospitalized patients
(20%), autoantibodies, drugs, passive
administration of other things like RhIG or IVIG
5) Also note that a negative DAT does not disprove an
acute hemolytic reaction
a) Donor RBCs all destroyed gives neg. DAT
page 2 Blood Bank IV P}Chaffin (2/11/2013)

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Figure 3: Direct Antiglobulin Test (DAT)

Image credit: A Rad 2006
d. Repeat ABO/Rh testing
1) Another check for right patient, right blood
2) Check both pre- and post-reaction specimens
5. Other things that may be done (but not required)
a. Repeat antibody screen (on both pre- and post-
transfusion samples
b. Repeat crossmatch with pre- and post samples
1) Best done with tube technique including immediate
spin and IAT phase readings +/- 37 C reading
c. Elution if DAT is + to determine specificity
d. Haptoglobin
1) Haptoglobin binds to free HGB molecules, cleared
by monocytes and macrophages in the RE system
2) Levels decrease sharply in acute intravascular
hemolysis (as well as extravascular)
3) Long turnaround time and acute phase reaction
make for limited usefulness in acute setting.
a) If you must use, compare pre- and post levels.
e. Direct and indirect bilirubin
1) Really more useful to confirm, not make diagnosis
2) Both will rise quickly, peak in less than 10 hours,
may be normal within 24 hours (if liver is OK)
f. Lactate dehydrogenase (LDH)
1) Abundant in RBCs (especially LD2 and LD1)
2) Not specific for intravascular hemolysis
g. Urine hemoglobin
1) Not as sensitive or as fast as hemoglobinemia
2) Hematuria does not equal hemoglobinuria!
c. Additional testing for suspected septic reactions:
1) Should be done if suggested by clinical data
a) Temperature greater than 102 F or >2 or 3oF
b) Severe rigors or other clinical findings
2) Both patient and product must be evaluated
a) Patient:
• Blood cultures
• Consider culture of all intravenous fluids

b) Product:
• Gram stain and culture of actual residual
product in the bag
d. Additional testing for suspected respiratory reactions
(see details in TRALI/TACO sections):
1) Chest X-ray
2) BNP levels
3) ABG
4) Donor testing for anti-HLA/HNA antibodies
e. Additional testing for suspected severe allergic
reactions:
1) Serum IgA levels (pretransfusion sample!)
2) Consider anti-IgA if serum IgA is non-detectable
C. Classification of reactions
Presenting With Fever

Acute Delayed
Acute Hemolytic Delayed Hemolytic
Febrile Non-hemolytic TA-GVHD
Transfusion-related Sepsis
TRALI
Presenting Without Fever
Acute Delayed
Allergic Delayed Serologic
Hypotensive Post-transfusion Purpura
Tx-associated Dyspnea Iron Overload
TACO
D. Acute reactions presenting with fever

1. Acute hemolytic transfusion reactions (AHTRs)
a. Incidence: 1:76,000 transfusions, (1:1.8 million
transfusions fatal HTR)
b. Clerical errors are most common cause
c. RBC destruction may be intravascular or extravascular
1) ABO-related, intravascular usually more severe
d. Signs/symptoms
1) Timing
a) Severe reactions may occur early in transfusion
(first 15 minutes; see figure 4)
b) Milder reactions may present later, but usually
before end of transfusion
2) Specific signs/symptoms:
a) Fever and chills
• Most common presenting symptom (> 80%)

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Figure 4
b) Back or infusion site pain
c) Hypotension/shock
d) Hemoglobinuria (1st indication anesthetized pts)
e) DIC/increased bleeding
f) Sense of “impending doom”
e. Lab findings
1) Hemoglobinemia (pink or red serum/plasma); lasts
several hours in those with adequate renal function
2) Hemoglobinuria (us clears by the end of one day)
3) Positive DAT (unless all donor cells destroyed);
may be “mixed field”
4) Elevated indirect and direct bilirubin
5) D-dimers, decreased fibrinogen, etc. (DIC)
6) RBC abnormalities
a) Schistocytes: Intravascular hemolysis
b) Spherocytes: Extravascular hemolysis
f. Pathophysiology
1) Intravascular hemolysis due to ABO incompatibility
typifies these reactions
a) ABO antibodies fix complement well and this
leads to rapid RBC lysis
b) Other antibodies (e.g., Kidd) may also fix
complement and lyse RBCs
c) Less commonly due to incompat. donor plasma
Figure 5: Classical Complement Pathway

Image credit: http://www.twiv.tv/classical-complement.jpg
2) Hemolysis leads to a complex array of events:

a) Release of free HGB and HGB-free stroma

b) Stimulation of intrinsic coag pathway and
bradykinin via Ag-Ab complexes
c) C3a and C5a generation (“anaphylatoxins”)
d) Production of several very important cytokines:
• TNF-, IL-1, IL-6, IL-8
Substance Effect
C3a/C5a Increases: Nitric Oxide (NO), cytokines,
histamine, leukotrienes
TNF- Increases: NO, tissue factor expression
Decreases: Thrombomodulin (anticoagulant;
assists protein C)
Interleukin-1 Increases: NO
(IL-1) Decreases: Thrombomodulin
Free HGB Scavenges NO (local, possible global decrease)
Bradykinin Transient hypotension
RBC Stroma Direct renal tubular damage
Table 1: Substances generated during acute HTRs and their effects
3) Net effects on various systems:
a) Inflammatory consequences:
• TNF-, IL-1, IL-6 strongly promote fever
• WBCs activated and stimulated by all
b) Coagulation consequences:
• Direct intrinsic path activation by Ag-Ab
complex interaction with factor XII
• Indirect activation of extrinsic path by TNF-
stimulation of tissue factor
c) Circulatory consequences:
• Increased C3a/5a, IL-1, TNF- stimulate
increased nitric oxide levels (vasodilation)
• Bradykinin from Ag-Ab complexes likewise
promotes transient systemic hypotension
d) Renal consequences:
• Sympathetic response leads to renal
vasoconstriction
• Free hemoglobin scavenges renal NO,
promoting vasoconstriction
• Renal microthrombi decrease renal flow
• HBG-free RBC stroma damages renal tubules
• Resultant oliguric renal failure in about 1/3 of
confirmed acute HTRs
e) Respiratory consequences:
• Anaphylatoxins promote histamine release,
with resultant wheezing/dyspnea
• Aggressive hydration during resuscitation
gives pulmonary edema risk
4) Extravascular hemolysis (e.g., Rh/Kell/Duffy, etc.)
is usually but not always less severe due to lack of
systemic complement and cytokine activation

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g. Treatment
1) Hydration/diuresis critical early components for
hypotension treatment and renal fx preservation
a) Urine output > 1 mL/Kg/hr with saline +/-
furosemide
b) Low-dose dopamine use is controversial
2) Consider DIC; (+/-) heparin
3) Consider early exchange transfusion, esp. for high-
volume incompatible transfusion
h. Prevention possibilities
1) Training and careful attention to phlebotomy,
labeling, issue, and administration
2) Two separate ABO/Rh types before transfusion
3) Advanced methods (RFID, bar codes, etc.)
2. Febrile nonhemolytic transfusion reactions (FNHTRs)
a. Historically most frequently reported reaction
1) Now 0.1 to <1% due to leukoreduction
2) Still more common than any reaction in heme-onc
and transplant patients, especially with PLTs
b. Unexplained increase in temperature of 1C or 2oF
1) Don’t require to diagnose or work up rxn
c. Cause: Increased pyrogenic substances (e.g., TNF-,
IL-1 ), mostly from WBCs
1) Cytokines produced before transfusion (e.g., PLTs)
• Donor WBCs secrete while in the storage bag.
• Transfusion infuses pre-formed substances
2) OR, cytokines made after transfusion (e.g., RBCs)
• Recipient anti-HLA/HNA antibodies attack
donor WBCs, or donor antibodies attack
recipient WBCs
• Leads to substance release after transfusion
d. Signs/symptoms
1) Transient fever/chills (+/- rigors?) during or up to 2
hours after transfusion
Figure 6
2) Symptoms usually later in transfusion (esp. PLTs)
3) Chills may be first; fever may be delayed up to one
hour or more after transfusion in up to 10% of cases
4) Premedicated or head injury patients may never
have fever

e. Differential diagnosis:
1) Acute HTR
a) Most AHTRs present with fever/chills alone
b) Impossible to distinguish early AHTR from
FNHTR clinically
2) Transfusion-related sepsis
a) Especially sepsis from PLT transfusions (RBC-
related sepsis usually presents earlier)
b) Timing may be identical
f. Lab findings
1) None (diagnosis of exclusion)
g Treatment
1) Antipyretics (acetaminophen)
2) Meperidine (Demerol) for more severe chills
h. Prevention
1) Acetaminophen premedication (doesn’t work well
as a routine prophylactic; use with repeat reactions)
2) Preventing FNH during RBC transfusions
a) Leukocyte reduction works extremely well to
prevent vast majority of these reactions
3) Preventing FNH during platelet transfusions
a) Bedside leukoreduction is ineffective;
substances are already in the bag!
b) Pre-storage leukoreduction best, but reactions in
~0.1 to 1% of PLT transfusions
3. Transfusion-related sepsis (septic transfusion reaction)
a. General statements
1) #1 infectious risk from transfusion
2) As many as 1 in 3000 platelet units are
contaminated (many fewer reactions, however)
b. How does it happen?
1) Platelets tend to be contaminated by skin
contaminants from collection process
2) RBCs more often contaminated by an organism
growing in the donor’s blood (often asymptomatic)
c. Organisms identified depend on product.
1) Red cells
a) Gram-negative rods (endotoxin-makers that like
growing in cold temperatures):
• Y. enterocolitica (most common historically)
• E. coli
• Enterobacter/Pantoea sp.
• Serratia marcescens and S. liquifaciens
• Pseudomonas species
b) Gram-positive organisms (much less commonly)
• Staph Epidermidis, Staph aureus
• Propionibacteria
2) Platelets
a) Vast majority are gram-positive cocci (skin)

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b) Gram neg rods less common but more likely to
cause fatalities (Serratia, E. coli, and Klebsiella)
3) Plasma products
a) Reports water bath contam. with Pseudomonas
d. Signs/symptoms
1) Earlier symptoms seen in more severe reactions and
more often with RBC transfusions (see fig 7)
2) Rapid onset high fever (often greater than 4oF/2C)
3) Rigors (true shaking chills with rigidity)
4) Abdominal cramping, nausea/vomiting
5) Hypotension/shock
6) DIC
e. Differential diagnosis:
1) Acute HTR (always!); Septic reactions usually more
“acute” and dramatic than the typical AHTR
2) Anaphylactic transfusion reaction: Similar
presentation but usually NOT febrile.
3) FNHTR: Milder septic reactions and many PLT
contaminations overlap with FNHTR (many culture
units as part of FNHTR workup protocol)
4) Sepsis from non-transfusion source (infected lines
and/or fluids, coincidental presentation)
Figure 7
f. Lab findings
1) Discolored RBC product (+/-); contaminated RBCs
may turn DARK or purple
2) May have hemoglobinemia/uria (non-immune)
3) DAT negative (unless coincidental)
4) Gram stain + in only half to 2/3 of proven cases!
a) Source of gram stain/culture is very important
b) Avoid culturing or staining a segment
c) Also culture associated IV fluids and consider
that an indwelling IV catheter as a source
5) Culture is proof positive (same organism cultured
from unit and recipient; better if from donor too!)
g. Treatment
1) Immediate IV antibiotics; treat presumptively with
broad spectrum coverage, then adjust as necessary
2) Pressure/respiratory/general support as needed
3) Notify blood collection agencies promptly!

h. Prevention
1) Careful donor history
2) Proper phlebotomy technique; diversion pouches
(divert contaminated skin plugs), strict attention to
possible site contaminants
3) Leukocyte reduction filters may decrease risk
(decrease in Yersinia concentration)
4) Routine detection of platelet contamination required
by AABB Standard
a) Culture-based methods (24 hrs post-collection):
• BacT/ALERT system: Microbiology standard
blood culture equipment
• Pall enhanced bacterial detection system
(eBDS): Detects bacterial use of oxygen
b) Pre-issue methods (shortly before issue)
• VeraxPGD system (LR apheresis PLTs and
pooled WBD PLTs); detects bacterial cell
wall antigens
• BacTx system (approved only for pooled
WBD PLTs)
5) Despite detection methods, false negatives occur,
and pathogen reduction may be the ultimate answer
4. Transfusion-related acute lung injury (TRALI)
a. #1 cause of transfusion-related fatality in the US!
1) Incidence varies: 1:1200 to 1:190,000 transfusions
b. Two almost identical standard definitions:
1) NHLBI Working Group and Canadian Consensus
Conference Panel
a) New acute lung injury < 6 hours post
transfusion; ALI defined:
• Hypoxemia with PaO2/FiO2 < 300 mm Hg
(or O2 sat <90%) and bilat CXR infiltrates
b) Lack of other risk factors for pulmonary edema
c) No pre-existing acute lung injury
2) Usually fever, chills, transient hypertension then
hypotension
3) PLTs/plasma transfusions, but also RBCs/WB
Figure 8
c. Clinical differential diagnosis:
1) ARDS: TRALI may look exactly like ARDS, but
TRALI usually resolves in 24-48 hours.

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2) Transfusion-associated circulatory overload
(TACO): TRALI usually febrile and does not
respond to diuretics
3) Anaphylactic reactions (generally afebrile)
4) Acute pulmonary and myocardial disorders
d. Pathophysiology: Two currently accepted pathways
1) The neutrophil is the villain in the pathophysiology!
a) Secretes toxic oxygen free radicals and other
substances damage endothelial cells, leads to
vascular leakage and TRALI
b) Almost 30% of PMNs are in lungs at all times!
2) Donor antibody pathway for TRALI (Figure 9)
Figure 9: Donor Antibody-mediated TRALI

(Image courtesy of Dr. Chris Silliman)
a) Anti-HLA or anti-neutrophil antibodies from
donor bind to antigens on recipient PMNs
b) Antibody-PMN complexes deposit in pulmonary
vasculature, activate PMN bactericidal response
c) Damage to pulmonary endothelial cells lining
capillaries, with resultant leakage of fluids into
the alveolar spaces (pulmonary edema)
d) This mechanism may also occur less commonly
with recipient antibodies against donor WBCs
e) Problem: Does not explain all TRALI reactions,
nor does it explain why TRALI does NOT occur
in settings where it SHOULD
3) “Two-event” pathway for TRALI (Figure 10)
a) First event: Pre-existing condition, activates lung
endothelial cells and primes PMNs
• Sepsis, major surgery, massive transfusion
b) Second event: Transfusion of stored blood
product (+/- antibodies)
• Stored blood products accumulate substances
called “biologic response modifiers” (BRMs)
that can prime/activate destructive PMNs

• Either BRMs or antibodies mentioned in the
first hypothesis may activate the primed
PMNs to secrete toxic substances
Figure 10: "Two-event" Synthesized Model for TRALI

(Image courtesy of Dr. Chris Silliman)
c) Combination of these events leads to capillary
damage and subsequent pulmonary edema.
d) This helps explain inconsistencies with the
donor antibody pathway and synthesizes the two
pathways nicely; still controversial though!
e. Diagnosis
1) Difficult, as it is often confused for something else
2) Typical early findings: Bilateral CXR infiltrates, O2
saturation < 90%, no JVD, normal wedge pressure
3) Lab: Anti-HLA and/or anti-PMN antibodies, +/-
increased BRM in the bag.
a) Clinical and radiographic diagnosis; confirming
the donor antibodies may take days or weeks!
f. Treat with respiratory support (O2, maybe intubation).
1) Mortality 5-25%, but 80% recover quickly
g. Prevention
1) AABB mandate to reduce TRALI risk
2) Implicated donors with antibodies deferred
3) Male-only plasma has been shown to decrease the
risk of TRALI (females have more anti-HLA and
anti-neutrophil antibodies because of pregnancy).
4) Some test female PLT donors for antibodies
5) Strategies ignore two hit model
E. Acute reactions presenting without fever
1. Allergic reactions
a. Mild allergic (urticarial, cutaneous) reactions
1) Very commonly reported reaction (1-3%)

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2) Localized hives, +/- more severe swelling around
eyes and lips (angioedema), mild respiratory
symptoms, and mild laryngeal edema
3) Mechanism
a) Type I (IgE-mediated) hypersensitivity to
transfused plasma proteins
b) Mast cell secretion of histamine and other
mediators of allergic reactions
4) Prevention and treatment options
a) Diphenhydramine IV 25-50 mg as treatment,
oral form as prophylaxis (not cost-effective)
b) Washed products work too (not usually done)
c) May restart transfusion after hives clear.
b. Moderate allergic reactions
1) Some reactions fall between the two extremes
2) May present with upper/lower airway obstruction
+/– cutaneous manifestations
a) Upper airway:
• Stridor, hoarseness, “lump” in throat
b) Lower airway:
• Wheezing, chest tightness, dyspnea
3) Some may respond to IV diphenhydramine, while
others will need epinephrine
c. Severe allergic (anaphylactic) reactions
1) Opposite end of hypersensitivity reaction spectrum
2) Uncommon (1:20,000 to 50,000 transfusions)
3) Presentation
a) Anaphylaxis very early in the transfusion
b) Acute hypotension, lower airway obstruction,
abdominal distress, systemic crash
c) Virtually all of these patients have skin
findings (urticaria, generalized pruritis)
4) What’s the allergen?
a) Classic: IgA deficient recipient with IgE anti-
IgA (induces severe type I reaction)
• Seen only in those with undetectable IgA
levels (i.e. IgA < 0.05 mg/dL)
• Problem: Labs don’t detect IgE anti-IgA!
• Vast minority of patients with anaphylactic-
type reactions have demonstrable IgA
deficiency with detectable anti-IgA
b) Haptoglobin deficiency in Asian patients; can
give same type of severe reaction as anti-IgA
c) Latex, drugs, foods in donors can lead to severe
reactions in susceptible recipients
d) Scattered reports of donors with IgE antibodies
transmitting temporary hypersensitivity
e) While all above are possible, very uncommon to
find specific reason for a severe allergic reaction

5) Differential diagnosis:
a) Acute HTR
• Typically febrile
• Most acute HTRs don’t present this early
b) Septic transfusion reaction
• High fevers, no skin findings in sepsis
• If unclear, give epinephrine anyway
c) Acute hypotensive reactions
• These reactions (see below) have hypotension
only, without respiratory or skin findings
6) Confirmation
a) ALL with a severe allergic reaction should have,
at minimum, check of pretransfusion IgA level
b) Those with very low/undetectable levels (<0.05
mg/dL) are tested for anti-IgA (detects IgG
antibody but could predict the possibility of IgE)
7) Prevention
a) IgA-deficient (IgAD) products given for those
with antibodies, but sometimes for low IgA only
• IgAD: Washed cellular products (RBCs,
PLTs), or products from IgA-deficient donors
• If possible, may also bank autologous units
b) Washed cellular products for those with severe
reactions and no demonstrable IgA deficiency
• MOST patients with history of severe allergic
reactions can get future untreated transfusions
without harm (monitor carefully however!)
c) Benadryl insufficient by itself; may use
corticosteroids +/- additional histamine blockers
8) Treatment
a) Epinephrine (0.2-0.5 ml of 1:1000 IM/SQ)
b) SQ or IM preferred, but IV ok if already crashed
2. Acute hypotensive reactions
a. Reactions similar to severe allergic reactions but no
skin symptoms, no GI or respiratory issues
1) CDC definition:
a) > 30 mm Hg drop in systolic BP; diastolic < 80
b) Occurs < 15 minutes after start of transfusion
c) Resolves < 10 minutes after transfusion stopped
b. Classically associated with two situations
1) Patients taking angiotensin-converting enzyme
inhibitors (ACEi)
a) Rapid onset of flushing and hypotension in
transfused patients who are on ACEi (e.g.,
Vasotec, Lotensin, Zestril, Capoten).
b) Probably accumulation of increased bradykinin
or metabolites during storage
• ACEi prevent bradykinin metabolism

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• Marked but transient hypotension (bradykinin
half-life is on the order of seconds
2) Receiving blood via negatively charged filters
a) Seen historically with certain negatively charged
bedside leukoreduction filters
b) Also in LDL apheresis and plasma exchange
with albumin replacement (esp. if on ACEi)
c) Reported with reinfusion of intraoperative blood
(cell saver) that is filtered before infusion
c. Diagnosis
1) Clearly a diagnosis of exclusion
2) Rule out:
a) Acute HTR by workup and lack of fever
b) Severe allergic reaction by lack of skin and
respiratory findings (as well as transience)
c) Septic reaction by lack of high fever, GI
complaints, and transience
d. Management
1) STOP the transfusion! (rapid resolution)
2) Give fluids, consider epinephrine if not resolved
e. Prevention
1) No routine prophylactic measures necessary
2) Avoid bedside leukoreduction filters (not really a
problem in most places)
3) Stop ACEi before therapeutic apheresis procedures
3. Transfusion-associated dyspnea (TAD)
a. A total garbage can diagnosis, in my view!
b. Definition:
1) Acute resp. distress < 24 hours after transfusion
2) TRALI, TACO, and allergy ruled out
4. Transfusion-associated circulatory overload (TACO)
a. Acute onset of congestive heart failure as a direct
result of blood transfusion
1) Dyspnea, orthopnea, bilateral rales, with hypoxia
2) Systolic hypertension (widened pulse pressure),
tachycardia, JVD, pedal edema, headache
3) Usually afebrile
4) X-rays with bilateral basilar infiltrates, widened
cardiac silhouette
5) Proposed diagnostic criteria include some of the
above signs/symptoms PLUS:
a) Hypoxemia (<90% saturation on room air)
b) Bilateral CXR infiltrates
c) Reaction occurring within 6 hours of transfusion
b. Patients most at risk (though any patient may get
TACO if transfused rapidly):
1) Patients with pre-existing CHF
2) Very old (>85% occur in patients over age 60) and
very young (to a lesser extent)

3) Renal failure
4) Chronic anemias due to compensation for anemia
with increased plasma volume
c. Differential diagnosis:
1) TRALI (see below)
2) Allergic/anaphylactic reactions
a) May present VERY early in transfusion (first
few drops)
b) Not responsive to diuretics or positional changes
3) Coincidental cardiac or pulmonary issues unrelated
to transfusion
a) Acute MI or pulmonary embolism
b) Valvular heart disease with decompensation
d. Distinguishing from TRALI
1) Clinical (response to diuretics/positional changes in
TACO, fever in TRALI)
2) CXR: Less cardiac silhouette widening in TRALI
3) Lab: Elevated brain natriuretic peptide (BNP)
suggests TACO (some use ratio of pre- to post-
transfusion >1.5 AND elevated post-transfusion)
4) Finding antibodies as above establishes TRALI
e. Treatment
1) Stop the transfusion, evaluate, sit patient up
2) Give supplemental oxygen
3) Diuretics to decrease blood volume
4) In severe cases, consider therapeutic phlebotomy
f. Prevention in at-risk patients
1) Control infusion rates (1 mL/Kg/hour).
2) Split units into aliquots when possible.
3) Consider lower volume units or volume reduction
F. Delayed reactions presenting with fever
1. Delayed hemolytic transfusion reactions (DHTRs)
a. Hemolysis occurring at least 24 hours but less than 28
days after transfusion (rare reports up to 6 weeks).
b. Pathophysiologic possibilities
1) Anamnestic response:
a) Patient exposed to non-self RBC antigen(s)
b) Antibody formed, but fades over time
c) Patient re-exposed to antigen in future
transfusion (antibody screen is negative)
d) Anamnestic rapid production of IgG antibody
e) Typical for Kidd, Duffy, Kell antibodies
2) Primary response
a) Patient exposed to non-self RBC antigen(s)
b) Antibody is formed quickly, and attacks still-
circulating transfused red cells
c) MUCH less common than anamnestic
c. Classically leads to extravascular hemolysis
1) IgG coats RBCs, removal in liver/spleen

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2) Peripheral smear will commonly show spherocytes
3) NOTE: DHTRs due to Kidd (Jk) antibodies may be
intravascular and severe (complement fixation)
d. Signs/symptoms
1) Often completely asymptomatic
2) Fever and anemia of unknown origin
3) Mild jaundice/scleral icterus may be seen
e. Lab findings
1) Icteric serum
2) DAT positive (classically “mixed field”)
3) Anemia
4) Newly identified RBC antibody
5) Spherocytes on peripheral smear
6) Elevated LDH and indirect bilirubin, decreased
haptoglobin (even if extravascular)
f. Treatment
1) As for AHTR if severe and intravascular
2) Often no treatment necessary
g. DHTR vs. “delayed serologic reaction” (DSTR)
1) Official definition of DSTR:
a) New, clinically significant RBC antibody in a
patient transfused > 24 hrs and < 28 days, AND:
b) Complete lack of evidence of hemolysis
2) Consider: Repeat antibody screen on pretransfusion
sample; evaluate bilirubin, haptoglobin, LDH,
peripheral smear, etc.
a) Any evidence of hemolysis in study above
changes the diagnosis from DSTR to DHTR
2. Transfusion-associated graft-vs-host disease (TA-
GVHD)
a. Results from an attack on recipient cells by viable T-
lymphocytes in a transfused blood product
b. TA-GVHD sequence/requirements:
1) Viable, active T-lymphocytes are transfused
2) Donor and recipient are not HLA-identical
3) Recipient is unable to respond to neutralize the
effect of the transfused WBCs
c. The normal response:
1) Transfused T-lymphocytes (CD4, CD8, and NK
cells) mount immune response vs foreign HLA host
2) Normally, host T-lymphs (CD8 and NK cells)
counterattack and neutralize the response (fig 11)
d. Lack of host neutralization (figure 12) may lead to
TA-GVHD, with continued T-lymph attack on host
1) Almost uniformly fatal, so thankfully rare
2) Patients present with:
a) Fever 7-10 days post-transfusion
b) Face/trunk rash that spreads to extremities
c) Mucositis, nausea/vomiting, watery diarrhea

d) Hepatitis
e) Pancytopenia and subsequent marrow aplasia
• Most patients die from infections
Figure 11: Normal Sequence
Figure 12: TA-GVHD

e. Radiation deactivates T-lymphs in transfused products
1) 2500 cGy (“rad”) dose required targeted to center of
bag, with at least 1500 cGy in all parts of the bag
2) Doesn’t significantly damage other cells
3) Why not leukocyte reduction?
a) Minimum threshold is not known
b) Reports of TA-GVHD from leukoreduced units
f. Patients potentially at-risk for TA-GVHD:
1) Immunosuppressed patients
a) Congenital T-cell deficiencies (DiGeorge’s,
SCID, Wiskott-Aldrich)
b) Stem cell or marrow transplant recipients
c) Patients taking chemo agents that attack T-cells
(Fludarabine, purine analogs)
d) Aplastic anemia patients
e) Patients with solid tumors getting intensive
chemotherapy/radiation
2) Intrauterine transfusions, premature neonatal
transfusions, and neonatal exchange transfusions

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3) Hematologic malignancies (esp. Hodgkin’s)
a) Inherent cellular defect in HD
b) Other heme malignancies at risk due to treatment
4) Patients with solid tumors and intense treatment
5) Granulocyte transfusion recipients
a) Fresh T-lymphs in short-shelf life product
6) Receiving blood from a first-degree relative
donor or receiving HLA-matched units
a) Specific: HLA-heterozygous recipient from an
HLA-homozygous donor (“One-way HLA
match”); see Figure 13 below
• Child 2 gets blood from child (child 1 HLA
homozygous, child 2 shares one haplotype)
• Child 1 sees child 2 as “non-self,” but child 2
does NOT see child 1 as “non-self” (no
counterattack)
Figure 13: One-way HLA Match

b) Occurs most frequently in families, but also in
less HLA-diverse populations (Japan)
d) Can lead to TA-GVHD in a completely
immunocompetent recipient
g. Patients probably NOT at risk:
1) Solid organ transplant recipients
2) Term neonates
3) AIDS patients (CD8 cells that counterattack
preserve function until late in disease).
4) Patients receiving previously frozen plasma
products (FFP, cryoprecipitate)
a) Disagreement over previously frozen RBCs need
for irradiation; no case reports of TA-GVHD
h. Don’t use irradiation for:
1) Preventing CMV transmission (leukocyte reduction)
2) Peripheral progenitor cell infusions (think about it)
i. Gamma irradiation and x-ray irradiation are used
interchangeably and are equally effective
j. Maximum storage: 28 days after irradiation or regular
expiration date, whichever comes first
1) K+ and free hemoglobin increase in plasma
G. Delayed reactions presenting without fever
1. Delayed Serologic Transfusion Reaction (DSTR)
a. Described above in the DHTR section

2. Post-transfusion Purpura (PTP)
a. Rare, with marked thrombocytopenia and increased
risk of bleeding about ten days following transfusion
(may be below 10,000/L)
1) Bleeding mucocutaneous (mouth and nose, GI
tract); intracranial hemorrhage < 10% of cases
2) Triggering transfusion platelets or RBCs
3) RBC products contain substantial amounts of
platelets and soluble platelet antigens
b. Multiparous females at risk (5:1 female-male ratio)
c. Caused by antibody vs common PLT antigen
1) Anti-HPA-1A (PLA1; 98% frequency) 70-80%
2) HPA-1A neg pts exposed via pregnancy/transfusion
3) HPA-1A-positive transfused platelets and HPA-
1a-negative patient platelets are both destroyed!
a) Antibody probably has autoantibody activity
b) Passive adsorption of Ag/Ab complexes or
soluble PLT Ags also suggested
d. Differential diagnosis is challenging and difficult
1) TTP, ITP, DIC, HIT all can share features
2) Even more difficult if already thrombocytopenic
d. IVIG normalizes platelet count in about 3-5 days
1) Use plasma exchange if IVIG fails only
2) Mortality 10% without treatment; now near 0%
with treatment.
e. Avoid platelet transfusion if possible
f. Future PLT transfusions negative for target antigen
3. Iron overload
a. Each unit of RBCs: 200-250 mg iron (generally, 1 mg
iron per 1 mL RBCs)
b. Lifetime load of ~50-100 transfusions in 70 Kg person
= risk for overload (big risk in chronically transfused)
1) Hepatic, cardiac, endocrine organ, RE system
deposition is especially damaging
2) May present with hepatic or cardiac failure,
diabetes, thyroid abnormalities
c. Exchange transfusions reduce risk
d. Iron chelators (deferoxamine, deferiprone, deferasirox)
remove iron from hepatic stores and from RE system
H. Consequences of significant reactions
1. FDA requirements
a. If there is suspicion that a death is transfusion-related,
FDA requires notification “as soon as possible” by
phone, fax, or e-mail (formerly 24 hours)
b. Full investigation and written report within 7 days
2. Joint Commission
a. AHTRs are “sentinel events” and require Root Cause
Analysis and reporting

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The Fun Just Never Ends…

page 2 Blood Bank I P}Chaffin (12/28/11)

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RBC Genotype Reaction with anti-Z

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2) As more A or B is made, less H remains.

Type Whites Blacks Asians Native Americans

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d. In secretors, Se product (FUT2) adds fucose, then Le

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5) Asians us. D+; their order is R1 > R2 > r and R0.

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c) Possible reasons for weak D

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b. Anti-M and anti-N can usually be ignored unless

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Blood Donation & Pretransfusion Testing

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Weight: > 110 lbs (50 Kg)

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A. Basic outline (see figure above)

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48. Have any of your relatives had Creutzfeldt- Permanent deferral

Medication Deferral List

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Blood Components and Component Therapy

Table 1: Storage Details for Various Blood Products

C. Quality control of blood products

Table 2: Quality Control for Blood Products (US)

II. Blood and Components

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9. Storage and shipping

b. May be pooled at blood center or transfusion service

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c. Why choose AD-PLTs over WBD-PLTs?

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