BLOOD BANK

LABORATORY PROCEDURES
WHAT IS BLOOD BANKING?
Blood banking is the process that takes
place in the lab to make sure that donated
blood, or blood products, are safe before
they are used in blood transfusions and
other medical procedures. Blood banking
includes typing the blood for transfusion and
testing for infectious diseases.
Facts about About 36,000 units of blood are needed every day.

blood banking
The number of blood units donated is about 13.6
million a year.

About 6.8 million volunteers are blood donors each

year.

Each unit of blood is broken down into components, such as red blood
cells, plasma, cryoprecipitated AHF, and platelets. One unit of whole
blood, once it's separated, may be transfused to several patients, each
with different needs.

Annually, more than 21 million blood components

are transfused.
WHO ARE THE
BLOOD DONORS?
Most blood donors are volunteers. However,
sometimes, a patient may want to donate blood
a couple of weeks before undergoing surgery,
so that his or her blood is available in case of a
blood transfusion. Donating blood for yourself is
called an autologous donation. Volunteer blood
donors must pass certain criteria, including the
following:

Must be at Must be in Must weigh Must pass

least 16 good at least 110 the
years of health pounds physical
age, or in and health
accordance history
with state exam given
law    before
donation
What tests are done in blood banking?
A certain set of standard tests are done in the lab once blood
is donated, including, but not limited to, the following:

Irradiation to blood cells is

Screening for performed to disable any T-
any unexpected lymphocytes present in the
red blood cell donated blood. (T-
antibodies that lymphocytes can cause a
may cause reaction when transfused, but
problems in the can also cause graft-versus-
recipient host problems with repeated
exposure to foreign cells.)

Screening for current or past infections,

• Typing: ABO Leukocyte-reduced blood has
including:
group (blood been filtered to remove the
Hepatitis viruses B and C
type) white blood cells that contain
Human immunodeficiency virus (HIV) antibodies that can cause
• Rh typing Human T-lymphotropic viruses (HTLV) I fevers in the recipient of the
(positive or and II transfusion. (These antibodies,
negative Syphilis with repeated transfusions,
antigen) West Nile virus may also increase a recipient's
Chagas disease  risk of reactions to subsequent
. transfusions.).
What are the blood types?
According to the American Association of
Blood Banks, distribution of blood types in the
U.S. includes the following:.

31% 9% 39% 3%

O Rh-negative - 9%
A Rh-negative - 6%
B Rh-negative - 2%
AB Rh-negative - 1%
 • Red blood cells. These cells carry oxygen to the
tissues in the body and are commonly used in the
treatment of anemia.
• Platelets. They help the blood to clot and are
used in the treatment of leukemia and other
forms of cancer.
• White blood cells. These cells help to fight
infection, and aid in the immune process.
• Plasma. is needed to carry the many parts of the
blood through the bloodstream. Plasma serves
many functions, including the following:
• Helps to maintain blood pressure
• Provides proteins for blood clotting
• Balances the levels of sodium and
potassium
• What are the components of • Cryoprecipitate AHF. The portion of the plasma
that contains clotting factors that help to control
blood? bleeding.
• While blood, or one of its components, • Albumin, immune globulins, and clotting factor
may be transferred, each component concentrates may also be separated and
processed for transfusions.
serves many functions, including the
following:
THANK YOU
Insert the Sub Title of Your Presentation
BLOOD BANK
Pre Transfusion Testing
WHAT TO OVERVIEW

EXPECT?
TESTING METHODS

REQUIRED COMPONENTS OF THE PROCESS

NOMENCLATURE
 A. Purpose
1. Provide a blood component to a patient that
will provide maximum benefit while minimizing
potential for harm.
2. This requires a systematic process that is
fairly rigid
3. Regulated in the US by:
a) CLIA (Clinical Laboratory Improvement
Amendments, 1988)
b) AABB (formerly “American Association of
Blood Banks”)
c) College of American Pathologists (CAP)
d) FDA

• Overview
 A. Purpose
1. Provide a blood component to a patient
that will provide maximum benefit while
minimizing potential for harm.
2. This requires a systematic process that is
fairly rigid
3. Regulated in the US by:
a) CLIA (Clinical Laboratory Improvement
Amendments, 1988)
b) AABB (formerly “American Association of
Blood Banks”)
c) College of American Pathologists (CAP)
d) FDA

• Overview
 A. Agglutination
1. Basic reaction in blood banking
2. Two stages:
a) Sensitization/coating
(1) Binding of antibody to surface of
RBCs
(2) Dependent on multiple factors
b) Bridge formation
(1) Linkage of adjacent RBCs that are
coated with antibody
(2) IgM is far more capable of forming
bridges between adjacent cells due to its
pentameric structure (total of 10 possible
bridging sites)
(3) IgG has a harder time, since it only
has two binding regions
• Testing principles
 A. Agglutination
3. Sensitization
a) Antigen + Antibody Antigen-Antibody
b) K0 = equilibrium constant of reaction
(1) Larger K0 means a push to the right
side of the equation, with more stable
and rapid reactions
c) Affinity of RBC antigens and antibodies
affected by multiple factors

(1) Cold-reactive (usually IgM) vs. warm-

reactive (usually IgG)
(a) Must react in appropriate
temperature for best antibody
detection
(b) Warm antibodies most important
• Testing principles (except ABO)
 A. Agglutination
(2) Size
(a) An RBC is over 700 times bigger than
an antibody!!!
(b) Overcoming this size difference in
vitro requires manipulation of
environment as well as forcing RBCs
closer together (centrifugation)

• Testing principles
 A. Agglutination
(3) Electrical repellence
(a) RBC surfaces have a negative charge
due to sialic acid at their surfaces
(b) RBCs are naturally repelled by the
negative charges (“zeta potential”)
and by the positively charged ionic cloud
that forms around the cells
(c) Reduce zeta potential with:
i) Low ionic strength solutions
(LISS) or albumin; fewer ions to
surround RBCs
ii) Water-exclusion (Polyethlyene
glycol, “PEG”)
(d) Zeta potential is one reason that IgG
molecules are challenged to directly
• Testing principles agglutinate RBCs (limited reach of
monomeric antibody)
i) RBCs usually don’t get closer
than about 14 nm apart
 A. Agglutination
(4) pH
(a) Optimal pH (7.0 in vitro) encourages
an environment where:
i) RBC surfaces are negatively
charged
ii) Antibodies are weakly positive
(b) Decreasing pH leads to
dissociation of antibody from RBC
surface
(5) Relative amounts of antibody and antigen
(a) Typical: 2 drops serum, 1 drop
RBCs
(b) This gives mild antibody excess,
to promote shift of equation to right
i) Too much antibody excess
• Testing principles can give “prozone” effect
(inhibiting agglutination), while
too much antigen excess
gives “postzone” effect (also
inhibiting agglutination)
 A. Agglutination
4. Direct vs. indirect agglutination
a) Direct agglutination
(1) Antibody binds to multiple RBCs and
causes agglutination without
additional manipulation
(2) IgM is much better able to do this than
IgG
(a) IgM maximum diameter is 30 nm
(vs 14 nm for IgG), which is wide
enough to more easily overcome
RBC zeta potential

(b) Occasional IgG antibodies can

do this, if antigens “stick up” fairly
far from RBC surface (ABO-related
• Testing principles & M and N antigens most
commonly)
 A. Agglutination
4. Direct vs. indirect agglutination

b) Indirect agglutination
(1) Antibody binds to, but does not form
bridges with, RBCs

(a) Requires additional step to see

agglutination (AHG phase
described
above; enzyme treatment of RBCs
may also make an IgG capable of
direct agglutination)
(2) Classically IgG rather than IgM, for
reasons mentioned above
• Testing principles (3) Most significant antibodies cause this
type of agglutination
1&2 - Give the two stages in Agglutination
3 -COLD REACTIVE ANTIBODY
4- WARM REACTIVE ANTIBODY
5,6,8 -GIVE 3 CARBOHYDRATE ANTIGENS
THAT IS FOUND IN RBCs
9,10,11 -GIVE 3 PROTEIN ANTIGENS THAT
IS FOUND IN RBCs
12&13 -TWO TYPES OF AGGLUTINATION
14 - AN ANTIBODY THAT HAS A
PENTAMERIC STRUCTURE
15 – REAGENT USED TO REDUCE ZETA
POTENTIAL
16,17,18,19,20 – GIVE 5 COMPONENTS
OF BLOOD

BONUS 20-25 GIVE 5 BLOOD SYSTEMS

OTHER THAN ABO&H SYSTEM.
BLOOD BANK
TUBE TESTING
 B. Tube Testing
Types of Blood Bank Reagents
1 . Reagent RBCs possess known antigens
and are treated to prolong their life span.
2. Antisera contain known antibodies against
specific RBC antigens.
3. Antiglobulin reagents contain poly- or
monospecific antibodies against human
antibodies.
4. Potentiators are solutions that enhance the
formation of antigen-antibody complexes.
Regulation of Reagent Production
1 . Blood bank reagents are licensed by the
Center for Biologies Evaluation and
Research of the Food and Drug Administration
(FDA).
• Overview 2. FDA specifies potency and specificity of
reagents before production.
 B. Tube Testing
Reagent Antisera
1 . Polyclonal: Many B cell clones produce
antibodies against antigens.
2. Monoclonal: A single B cell clone produces
antibody against an antigen.
a. Advantages: Endless production,
exactly the same reagent in each batch,
no human/animal sources, no
contamination
b. Disadvantages: Single specificity; may
not react with all portions of RBC
antigen
3. Blended monoclonal: Reduces
disadvantages of single clone
• Overview
 B. Tube Testing
Reagent Antisera
4. ABO antisera
a. Anti-A and Anti-B reagents are used to
determine if patient is A, B, AB,
or O.
b. Anti-A reagent is colored with a blue
dye; Anti-B reagent is colored
with a yellow dye.
c. Suspension of patient's cells is added
to antisera.
d. Agglutination read at immediate spin.
These antibodies are IgM and react
best at room temperature or 4°C.

• Overview
 B. Tube Testing
Reagent Antisera
D typing
a. These are important antigens to detect
because antibody-antigen
reactions in vivo cause HTR and HDN.
b. Two types of reagents:
1) High protein: Older reagent; need to
run an Rh control with this
reagent because the protein in the diluent
may cause false positive
reactions in patients with autoantibodies
or abnormal serum proteins.
2) Low-protein monoclonal: Rh control is
usually not required. Only
need to perform a control when patient
• Overview has abnormal serum proteins.
 B. Tube Testing
Antiglobulin reagents
a. Polyspecific: Detects both anti-IgG and anti-
C3; used often in direct antiglobulin tests (DAT)
b. Monoclonal: This may be used to
differentiate between antibodies to IgG and C3.
Monospecific (antihuman IgG) reagents are
typically employed
for tests requiring an antiglobulin phase of
testing.
Reagent RBCs
a. IgG-coated control cells (Check cells): AABB
Standards for Blood Banks and Transfusion
Services require a control to ensure antiglobulin
reagent reactivity in each negative antiglobulin
test tube. Check cells are prepared by
• Overview attaching an IgG antibody to RBCs (sensitized
RBCs).
 B. Tube Testing
b. Aj and B cells for reverse grouping: These
are used to confirm front typing results. These
cells detect ABO antibodies. Landsteiner's Law:
If the patient's RBCs have an antigen, they do
not have the antibody in the serum.
c. Antibody screening cells: These are used to
detect antibodies present in a patient's serum.
Antibodies must be detected before patients
are
transfused to prevent hemolytic transfusion
reactions and/or death. Each set of screening
cells has two or three antigenically different
RBC reagent red cells. The antigens of each
cell are known and printed on an antigram
included with each set.
• Overview d. Antibody panel cells: Antibody identification
procedures use panel of RBCs whose antigens
are known. The panel consists of 10 to 20 vials
of these RBCs. Every panel has an antigenic
profile that lists all of the known
 B. Tube Testing
e. Other methods for antigen-antibody reaction detection
1) Gel technology: This technique uses dextran
acrylamide gel combined
with reagents or diluent. Anti-IgG cards are used for
DATs and lATs.
2) Microplate methods: The traditional tube method
is adapted to the microtiter plate, in which smaller
volumes of serum and cells are
used, and it is read on an automated photometric
instrument. The cell buttons are resuspended by
tapping the sides of the plate.
3) Solid-phase adherence methods
a) RBC screening cells are bound to surface of
microtiter plates.
b) Add patient's serum.
c) RBCs capture IgGs.
d) Plates washed
• Overview e) Indicator cells (anti-IgG-coated RBCs) are in
contact with bound
antibody.
f) Negative = RBC button; Positive = RBCs (indicator
cells) on sides and bottom of wells
 B. Tube Testing
4) Indirect antiglobulin test (IAT)
a) Purpose of the IAT is to detect in vitro
sensitization of RBCs.
b) In this procedure, reagent red cells are
mixed with patient's serum,
then incubated at 37°C to allow IgG
antibodies to attach to the RBCs. The
solution is then washed to remove
unbound proteins. AHG is added to detect
in vitro sensitization of RBCs.
c) False positive tests result from RBCs
being agglutinated before the washing
step (cold agglutinin), improper RBC
suspension, dirty
glassware, and overcentrifugation.
• Overview d) False negative tests result from poor
washing of RBCs, testing being delayed,
loss of reagent activity, no AHG added, or
use of an
improper RBC suspension
 B. Tube Testing
5) Potentiating media (antibody enhancers)
a) Definition: Reagents added to the in vitro
antiglobulin test to enhance antigen-antibody
complex formation
b) LISS increases antibody uptake of antigen.
c) Bovine albumin (22% or 30%) allows
sensitized cells to come close together to form
agglutination lattices.
d) Polyethylene glycol (PEG) additive
concentrates antibodies and creates a low-ionic
solution to allow greater antibody uptake.
e) Proteolytic enzymes: Papain, ficin, and
bromelin are used. Enzymes remove certain
structures from the RBC and enhance
the access of antibodies to other less
• Overview superficial structures on the RBC. Antibodies
that are enhanced include Rh, Kidd, and Le
blood group systems. The following antigens
are destroyed by the enzymes: M, N, S, Xga,
Fya, and Fyb.
 B. Tube Testing
1. Three main “phases”
a) Immediate spin (IS)
(1) 2 drops serum + 1 drop of 2-5% RBC
solution, centrifuge 15-30 sec
(2) Detects IgM antibodies best
(3) Antibodies seen at this phase only are
usually not clinically significant
b) 37 C
(1) Add potentiator, incubate at 37 C
(see table), centrifuge
(2) Occasional cold-reacting IgM
antibodies and occasional warm-reacting
IgG antibodies react at 37 C; in short, it’s
not that useful by itself
(3) Some do not perform a 37 C read, as
• Overview it doesn’t add much in most cases
(a) NOTE: Those using PEG potentiation
should NOT do a 37 C read, as
PEG can induce nonspecific positive
reactions that are meaningless
 B. Tube Testing
c) Antihuman globulin (AHG)/Indirect antiglobulin
test (IAT)
(1) AHG is antibody vs. human antibodies and/or
complement
(2) Wash same tube as used for 37 C, add AHG,
centrifuge
(a) Washing removes unbound globulins that
“neutralize” (are bound by)
AHG and cause false negative reactions
(3) Only required part of antibody detection, because
it is best for detecting
warm-reacting IgG antibodies
(4) Polyspecific or IgG-specific AHG may be used
(lab preference)
d) Grading reactions
(1) Read after gentle resuspension of button (micro
exam not necessary)
• Overview (2) Generally on a 0-4+ scale (some major labs use
0-12 scale instead)
(3) Negative: Smooth, easily dispersed RBCs
(4) Strong positive (4+ or 12): Tight cell button, not
easily dispersed
(5) Gradually increasing agglutinates from 1+ to 3+
 B. Tube Testing
c) Antihuman globulin (AHG)/Indirect antiglobulin
test (IAT)
(1) AHG is antibody vs. human antibodies and/or
complement
(2) Wash same tube as used for 37 C, add AHG,
centrifuge
(a) Washing removes unbound globulins that
“neutralize” (are bound by)
AHG and cause false negative reactions
(3) Only required part of antibody detection, because
it is best for detecting
warm-reacting IgG antibodies
(4) Polyspecific or IgG-specific AHG may be used
(lab preference)
d) Grading reactions
(1) Read after gentle resuspension of button (micro
exam not necessary)
• Overview (2) Generally on a 0-4+ scale (some major labs use
0-12 scale instead)
(3) Negative: Smooth, easily dispersed RBCs
(4) Strong positive (4+ or 12): Tight cell button, not
easily dispersed
(5) Gradually increasing agglutinates from 1+ to 3+
 B. Tube Testing
C. Column agglutination (“gel”) testing
1. Essentially “skips” the IS phase,
incorporates the 37 C incubation into
the process, and gives an AHG result
2. Performed in microtubes with a
chamber at the top for mixing plasma and RBCs
and a column filled with gel and anti-IgG a) Add
plasma and RBCs to chamber at top, incubate at
37 C, centrifuge
3. Gel separates agglutinates based on size and by
binding to IgG-coated RBCs
a) Strong positive gives agglutinates at the
TOP of the gel
b) Complete negative gives RBCs at the
BOTTOM of the gel
• Overview c) 4. Like PEG-enhanced tube testing,
excellent for detecting warm antibodies
(and has similar sensitivity)
B. Tube Testing
D. Solid phase testing (see diagram at right; courtesy of
Immucor)
1. RBC antigens are bound to microplate wells
(either on intact or lysed RBC membranes), then
plasma added, with incubation at 37 C
a) If IgG against an RBC antigen(s) is present,
antibody binds to antigen all over the bottom
of the well
2. Wash away unbound antibodies, then add RBCs
coated with anti-IgG, which bind to the
previously attached IgG on the bottom of the well
3. Positive and negative are opposite of what you
might think
a) Negative: Solid “button” in the bottom of the
well (indicating that there were no attached
plasma antibodies with which the anti-IgGcoated
indicators cells could bind)
b) Strong positive (4+): Diffuse “carpet” of
indicator RBCs spread all across the bottom
of the well (indicating that the plasma
antibody is attached to the well-bound RBC
antigens)
4. Sensitivity basically equivalent to gel and PEG
methods
B. Tube Testing
E. Indirect antiglobulin test (IAT)
1. A test to detect in-vitro coating of RBCs with
antibody and/or complement
2. Serves as the third phase of tube testing, and is
the main part of the antibody screen
3. Procedure:
a) In pretransfusion testing, patient serum
added to solution of donor RBCs, to
check for incompatibility between recipient
antibodies and donor RBCs

b) Can be done with known serum and

antibody and unknown RBCs, or with
RBCs of a certain phenotype to check for
serum antibody
B. Tube Testing
4. Types of anti-human globulin (AHG)
a) “Polyspecific” (polyclonal anti-IgG + monoclonal
anti-C3d)
(1) Previously the most commonly used AHG,
but less popular now
(2) If positive, labs would then do tests with
anti-IgG and anti-C3 individually
b) Anti-IgG
(1) Used for gel and solid-phase platforms
exclusively
(2) Many labs use anti-IgG only in tube tests,
as well
(3) Can get some cross-reactivity with other
immunoglobulin types due to reaction with
kappa and lambda light chains shared by
immunoglobulins
B. Tube Testing
c) Anti-C3d
(1) C3d is a nonreactive byproduct of
complement fixation on RBCs
(2) Anti-C3d is useful for evaluating IgM-
related hemolysis and cold agglutinin
disease, where antibodies are not usually
detectable via anti-IgG
5. AHG control (“check cells”)
a) For all negative tube IAT or DATs, add reagent
RBCs coated with antibody and/
or complement; should see free AHG agglutinating
the check cells
(1) No agglutination means test or reagent
problem
b) Gel-negative IAT/DAT tests do NOT require an
additional AHG control
c) Solid-phase tests run a positive control in
parallel, so no additional AHG control
required
B. Tube Testing
F. Direct antiglobulin test (DAT)
1. Detects whether antibody or complement coating
of RBCs has occurred in vivo
2. Procedure:
a) Essentially just the last part of performing
an IAT.
3. DATs are useful in workup of:
b) Transfusion reactions
c) Autoantibodies and autoimmune
hemolytic anemia
d) Hemolytic disease of the fetus/newborn
e) Drug-related hemolytic anemia
f) Antibodies vs. recently transfused
antigens
4. Positive DATs, however, are nonspecific, and are
seen in up to 15% of hospitalized patients
B. Tube Testing
G. A few other tools in the pretransfusion testing
portfolio:
1. Proteolytic enzymes
a) Cleave proteins on RBC surface, may
make underlying antigens more available
b) Destroys some antigens (e.g., Duffy and
MNS), enhances others (ABO, Rh, Jk)
c) Not used routinely, but often used in
complex or difficult cases
2. Prewarming
a) Performing pretransfusion testing with all
reagents and samples incubated and
kept at 37 C can help eliminate effects of cold
auto- or alloantibodies
b) NOT to be used as a way to get rid of
reactivity of stuff you don’t understand!
(1) The procedure may weaken some significant
antibodies
(2) Should be used only as confirmation of the
workup you have already done
B. Tube Testing
G. A few other tools in the pre transfusion testing
portfolio:
3. Adsorption
a) Removal of specific antibodies from
sample via incubation with antigen positive
RBCs
b) Used to remove warm or cold
autoantibodies (“autoadsorption”) from
sample in order to detect underlying
alloantibodies
c) May also be used to remove one or more
alloantibodies (“alloadsorption”) from sample
in order to detect or confirm the presence of
other alloantibodies
(1) May be used with multiple
antibodies to help clear a muddy picture
(2) e.g., Sample has anti-K, anti-C, and
anti-S but anti-S isn’t visualized well.
Use K+C+S– RBCs to adsorb the anti-K
and anti-C and leave the anti-S in
the “adsorbed serum” for clearer
B. Tube Testing
G. A few other tools in the pretransfusion testing
portfolio:
4. Elution
a) Technique for removal of antibodies bound to RBC
surface for analysis
b) May be done with heat, cold, chemical (e.g.,
glycine) treatment
5. Other RBC treatments
a) Dithiothreitol (DTT) or 2-mercaptoethanol (2-ME)
(1) Denatures surface RBC antigens of multiple
groups (including Kell,Lutheran, Dombrock, Yt, LW)
(2) Can also be used to remove IgM antibody activity
from serum
b) ZZAP
(1) Combination of DTT and proteolytic enzyme
(2) Acts on combination of enzyme sensitive and
DTT-sensitive antigens
c) Chloroquine
(1) Removes IgG from coated (DAT-positive) RBCs to
allow for accurate phenotyping (effective at least 80%
of the time)
(2) Also removes residual HLA antigens from RBCs
(Bg antigens)
B. Tube Testing
1. The antiglobulin test does not require washing
or the addition of IgG-coated cells in which of the
following antibody detection methods?
A. Solid-phase red cell adherence assays
B. Gel test
C. Affinity column technology
D. Polyethylene glycol (PEG) technique
2. What is the process of removing an antibody
from the red blood cell membrane called?
A. Absorption
B. Adsorption
C. Elution
D. Immunization
 3. Which of the following tests on donor red
blood cells must be repeated by the transfusing
facility when the blood was collected and
processed by a different facility?
A. Confirmation of ABO group and Rh type of
blood labeled D-negative
B. Confirmation of ABO group and Rh type
C. Weak D on D-negatives
D. Antibody screening
 4. Most blood group antibodies are of what
immunoglobulin classes?
A. IgA and IgD
B. IgAand IgM
C. IgE and IgD
D. IgGand IgM
 5.What type of transfusion reaction is often
diagnosed by a positive DAT and a gradual drop
in the patient's hemoglobin level?
A. Anaphylactic
B. Febrile
C. Delayed hemolytic
D. Acute hemolytic
6-9 Give the three main phases of tube testing
10. Test that detects whether antibody or complement
coating of RBCs has occurred in vivo.
11-14 Types of Blood Bank Reagents
11 . _______________ possess known antigens and are
treated to prolong their life span.
12. _______________contain known antibodies against
specific RBC antigens.
13. _______________contain poly- or monospecific
antibodies against human antibodies.
14. _______________are solutions that enhance the
formation of antigen-antibody complexes.
15-19 Fill in the Blanks

20. A test to detect in-vitro coating of RBCs with antibody

and/or complement