COLLEGE OF HEALTH – HAIL
Medical laboratory Dept.- Second term
THIRD YEAR – Blood banking
PRACTICE -4
Antibody Detection,
Antibody Identification
Indirect Antiglobulin Test (IAT) for the Detection of Antibodies
to Red Cell Antigens
Specimen
Serum or plasma may be used. The age of
the specimen must comply with pretransfusion
specimen requirements in AABB
Standards for Blood Banks and Transfusion
Services.
Reagents
1. Normal saline.
2. Bovine albumin (22% or 30%).
3. LISS made as follows:
a. Add 1.75 g of NaCl and 18 g of glycine to a 1-liter volumetric flask.
b. Add 20 mL of phosphate buffer prepared by combining 11.3 mL
of 0.15 M KH2PO4 and 8.7 mL of 0.15MNa2HPO4.
c. Add distilled water to the 1-liter mark.
d. Adjust the pH to 6.7 ± 0.1 with NaOH.
e. Add 0.5 g of sodium azide as a preservative. Note: LISS may be used
as an additive or for the suspension of test red cells. LISS preparations
are also available commercially.
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4. PEG, 20% w/v: To 20 g of 3350 MW PEG, add phosphate-buffered
saline PBS) pH 7.3 (seeMethod 1.7) to 100 mL. PEG is also available
commercially.
5. Antihuman globulin (AHG) reagent. Polyspecific or anti-IgG may be
used unless otherwise indicated.
6. Commercially available group O antibody detection cells. Pooled
group O antibody detection cells may be
used only for donor testing. Testing of patients’ samples must be
performed
with unpooled cells.
7. IgG-coated red cells.
Albumin or LISS-Additive Indirect Antiglobulin
Test Procedure
1. Add 2 drops of serum or plasma to
properly labeled tubes.
2. Add an equivalent volume of 22% or
30% bovine albumin or LISS additive
(unless the manufacturer’s directions
state otherwise.)
3. Add 1 drop of a 2% to 5% saline-suspended
reagent or donor red cells to
each tube and mix.
4. For albumin, incubate at 37 C for 15
to 30 minutes. For LISS, incubate for
10 to 15 minutes or follow the manufacturer’s
directions.
5. Centrifuge and observe for hemolysis
and agglutination. Grade and record
the results.
6. Perform the test described in Method
3.2.1, steps 6 through 9.
Interpretation (for Antiglobulin Tests,
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1. The presence of agglutination/hemolysis
after incubation at 37 C constitutes
a positive test.
2. The presence of agglutination after
addition of AHG constitutes a positive
test.
3. Antiglobulin tests are negative when
no agglutination is observed after
initial centrifugation and the IgGcoated
red cells added afterward are
agglutinated. If the IgG-coated red
cells are not agglutinated, the negaPrewarming Technique Principle
Prewarming may be useful in the detection
and identification of red cell antibodies
that bind to antigen only at 37 C. This
test is particularly useful for testing sera
of patients with cold-reactive autoantibody
activity that may mask the presence of
clinically significant antibodies. However,
use of the prewarming technique for this
application has become controversial.1-2 It
has been shown to result in decreased
reactivity of some potentially significant
antibodies and weak antibodies can be
missed.3 The technique should be used
with caution and not used to eliminate
unidentified reactivity.
Strong cold-reactive autoantibodies may
react in prewarmed tests; other techniques
such as cold allo- or autoadsorption or
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dithiothreitol treatment of plasma may be
required to detect underlying clinically significant
antibodies.
Specimen
Serum or plasma may be used. The age of
the specimen must comply with pretransfusion
specimen requirements in AABB
Standards for Blood Banks and Transfusion
Services
Reagents
1. Normal saline.
2. Anti-IgG.
3. Commercially available group O antibody
detection cells. Pooled group
O antibody detection cells may be used
only for donor testing. Testing of patients’
samples must be done with
unpooled cells.
4. IgG-coated red cells.
754 AABB Technical Manual
Procedure
1. Prewarm a bottle of saline to 37 C.
2. Label one tube for each reagent or
donor sample to be tested.
3. Add 1 drop of 2% to 5% saline-suspended
red cells to each tube.
4. Place the tubes containing red cells
and a tube containing a small volume
of the patient’s serum and a pipette
at 37 C; incubate for 5 to 10 minutes.
5. Using the prewarmed pipette, transfer
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2 drops of prewarmed serum to
each tube containing prewarmed red
cells. Mix without removing tubes
from the incubator.
6. Incubate at 37 C for 30 to 60 minutes.
7. Without removing the tubes from the
incubator, fill each tube with prewarmed
(37 C) saline. Centrifuge
and wash three or four times with 37
C saline.
8. Add anti-IgG, according to the manufacturer’s
directions.
9. Centrifuge and observe for reaction.
Grade and record the results.
10. Confirm the validity of negative tests
by adding IgG-coated red cells.
Notes
1. The prewarming procedure described
above will not detect alloantibodies
that agglutinate at 37 C or lower and
are not reactive in the antiglobulin
phase. If detection of these antibodies
is desired, testing and centrifugation
at 37 C are required. If time permits,
a tube containing a prewarmed
mixture of serum and cells can be
incubated at 37 C for 60 to 120minutes,
and the settled red cells examined
for agglutination by resuspending
the button without centrifugation.
2. Cold-reactive antibodies may not be
detectable when room-temperature
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saline instead of 37 C saline is used
in the wash step.2 The use of roomtemperature
saline may avoid the
elution of clinically significant antibody(
ies) from reagent red cells that
can occur with the use of 37 C saline.
Some strong cold-reactive autoantibodies,
however, may still react and
therefore require the use of 37 C saline
to avoid their detection.
Saline Replacement to Demonstrate
Alloantibody in the Presence of Rouleaux
Principle
Rouleaux are aggregates of red cells that,
characteristically, adhere to one another
on their flat surface, giving a “stack of coins”
appearance when viewed microscopically.
Rouleaux formation is an in-vitro phenomenon
resulting from abnormalities of
serum protein concentrations. The patient
is often found to have liver disease,
multiple myeloma, or another condition
associated with abnormal globulin levels.
It may be difficult to detect antibody-associated
agglutination in a test system
containing rouleaux-promoting serum. In
the saline replacement technique, serum
and cells are incubated to allow antibody
Procedure
1. Prewarm a bottle of saline to 37 C.
2. Label one tube for each reagent or
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donor sample to be tested.
3. Add 1 drop of 2% to 5% saline-suspended
red cells to each tube.
4. Place the tubes containing red cells
and a tube containing a small volume
of the patient’s serum and a pipette
at 37 C; incubate for 5 to 10 minutes.
5. Using the prewarmed pipette, transfer
2 drops of prewarmed serum to
each tube containing prewarmed red
cells. Mix without removing tubes
from the incubator.
6. Incubate at 37 C for 30 to 60 minutes.
7. Without removing the tubes from the
incubator, fill each tube with prewarmed
(37 C) saline. Centrifuge
and wash three or four times with 37
C saline.
8. Add anti-IgG, according to the manufacturer’s
directions.
9. Centrifuge and observe for reaction.
Grade and record the results.
10. Confirm the validity of negative tests
by adding IgG-coated red cells.
Notes
1. The prewarming procedure described
above will not detect alloantibodies
that agglutinate at 37 C or lower and
are not reactive in the antiglobulin
phase. If detection of these antibodies
is desired, testing and centrifugation
at 37 C are required. If time permits,
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a tube containing a prewarmed
mixture of serum and cells can be
incubated at 37 C for 60 to 120minutes,
and the settled red cells examined
for agglutination by resuspending
the button without centrifugation.
2. Cold-reactive antibodies may not be
detectable when room-temperature
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