Elution and Adsorption Techniques

Practical session no 11
Done by nada fallatah ,2016

Elution Techniques:
 It is the process of removing antibodies from the surface of

red blood cells. It is performed, when the DAT is positive
with IgG antibody or complement.
 Antibody release (elution) can be accomplished either

by:
1- Disrupting the linkage between antigen and antibody.
2- Changing conditions that favor dissociation of antibodies

from the antigen.
1- The Types Of Elution Techniques:
1- Acid elution (glycine acid):
 Used for eluting warm-reactive auto- and alloantibodies.
 Most common.
 Lowers pH, causing antibody to dissociate.
2- Organic solvents (ether, chloroform):

 Dissolve bilipid layer of RBC.
3- Heat or freeze-thaw elution techniques:

 Used for investigation of HDN caused by ABO incompatibility.
 Heat (conformational change).
 Freeze-Thaw (lyses cells).

2- Intended use:
The purpose of an elution is releasing the antibody from the
surface of RBCs.
Once they are free in the solution, the eluted antibodies are
tested against reagent red blood cells (panel cells) to determine
if there is an immune antibody there or not.
Positive DAT
Y Free antibody Antibody ID
Elution
Sensitized
Y
Y
RBC
Y
Y
3- Principle of Heat Elution:
1-Heat elution is best suited for :
1-The investigation of ABO hemolytic disease of the
newborn.
2- Elution of IgM antibodies with patient who have been
transfused with non-ABO-identical blood.
2-Six to eight times wash the coated red blood cells with
large volumes of saline to remove all antibodies except that
bound to the cells.
3- An albumin solution is added and the cells are heated to

56 oC. Results in, cause disruption of the antigen-antibody
bond; the antibody will “pop off” the coated RBCs into the
albumin solution.
4- Centrifuged the solution and the supernatant fluid
(eluate) is removed.
5- The supernatant fluid contains the antibodies that

were coating the cells.
The eluate is tested against screening and panel cells to

determine the antibody specificity.
4- Sample:
Cord Blood Sample Positive for DAT.
5- Method of heat elution procedure:

1- Centrifuge the sample with the positive DAT.
2- Separate the(serum or plasma) properly in labeled tube.
3- add 20 drops of the red cells in a properly in a new labeled
tube.
4- Wash the cells 6- 8 times with large volumes of saline.
5- Mix cells vigorously between washes by adding a low
volume of saline and covering the tube with Para film and
inverting the tube until all cells are re-suspended. And so,
complete filling of tube with saline.
6- Add 20 drops of 6% bovine albumin to the washed packed
red blood cells.
7- Place the tubes at 56°C for 10 minutes, agitating the tube
periodically during this time.

8- Centrifuge the tube at (3000 RPM) for five minutes.
9-Immediately transfer the supernatant (eluate) into a clean
test tube, and test in parallel with the supernatant (saline)
from the final wash.
6-Interpretation - Eluate Testing For ABO Antibodies:
1- If the last wash is positive with any of the test cells. the
eluate indicates present of antibodies specific for the antigen
on the test cells.
2- Lack of agglutination of test cells by the eluate indicates

absence of antibodies specific for the antigens on the test
cells.
7- Diagnostic significance:
1. Investigation of HTR.
2. Diagnosis of autoimmune hemolytic anemia (AIHA).
3. Diagnosis ABO hemolytic disease of the newborn (HDN).
4. Identification of specificity when multiple antibodies exist
in a patient's serum.
5. Phenotyping red cells in patients with a positive DAT.
Adsorption Techniques
 Adsorption is removal of antibody from a plasma/serum by

incubating it with red cells have the corresponding antigen.
 Adsorption procedures can be used to investigate underlying

alloantibodies.
 ZZAP or chloroquine diphosphate can be used to dissociate

IgG antibodies from the RBC. After the patient RBCs are
incubated, the adsorbed serum is tested with panel cells to ID
the alloantibody (if present).
 Adsorption are most commonly used (when a serum
sample contain multiple anti body) to remove auto
antibody because it interfere with antibody identification
of alloantibody due they react with all reagent and donor
cells.
There are two types of adsorption and depending on the
transfusion status of the patient, when DAT is positive.
1-Auto-adsorptions: use the patient's own RBCs to remove
the autoantibody from the patient's serum without
removing any alloantibodies (it is done for the patient that
HAS NOT BEEN transfused or pregnant in the last 13
weeks).
 Warm auto adsorption : performed at 37 and used to
remove IgG autoantibodies.
 Cold auto adsorption : performed at 4 and used to
remove IgM auto antibodies.
2- Allo- adsorptions: use RBCs of known phenotypes to
selectively remove alloantibodies from the patient's serum
(it is done for the patient that HAS BEEN transfused).
1-Principle:
Mix serum with antibody with red cells with corresponding
antigen. Incubate at appropriate temperature. Anti-e antibodies
have been adsorbed out of the serum and onto the RBCs.
Anti-e Anti-e Anti-S

Anti-e
Anti-e
Anti-S Anti-e
Anti-e
Anti-e
Anti-S Anti-S
2: sample:
Serum or plasma containing antibody to be adsorbed.
3:Reagents:
Red cells of panel for identification that carry the antigen

corresponding to the antibody specificity to be adsorbed.
4- Method of Auto-adsorptions:
1- Prepare ZZAP reagent by mixing 0.5 mL of 1% cysteine-
activated papain with 2.5 mL of 0.2 M DTT and 2 mL of pH
7.3 PBS.
2- To each of two tubes containing 1 mL of red cells, add 2
mL of ZZAP reagent. Mix and incubate at 37 C for 30 minutes
with periodic mixing.
3- Wash the red cells three times in saline. Centrifuge the
last wash for at least 5 minutes at (3000 rpm) and remove as
much supernatant saline as possible.
4- Incubate for 30-45 minute at 4 oC or 37 oC, depending
upon the type of antibody.
5- Mix the tube every 10 minutes by inverting the tube until
all cells are re-suspended off the bottom of the tube.
6- Centrifuge for 5 minutes at 3400 rpm and carefully move
the serum and put it in the second aliquot of ZZAP treated
cells and repeat steps 3to 6 to adsorb the auto out of the
plasma.
7- Test adsorbed plasma again against antibody panel cells to
detect and identify alloantibodies.
Note: As a guide, when the original serum reactivity is
2+ to 3+ reactivity will generally be removed in two to
three adsorptions. Performing greater than four
adsorptions increases the risk of diluting alloantibody
reactivity.
5- Results Interpretation:
1- If reactivity remains, the antibody has not been
completely removed. Carefully move the adsorbed serum
and put it in the NEW aliquot of ZZAP treated cells and
repeat steps 3 to 6 to adsorb the auto out of the plasma
2- If no reactivity, that means antibody has been completely

adsorbed. Test adsorbed plasma against panel cells to
identify alloantibodies
6- Diagnostic significance:
1) Removing autoantibody to permit detection and
identification of possible underlying coexisting
alloantibody(ies) is the main reason why we send cases to the
Immunohematology Reference Lab to perform adsorption
techniques.
2) Reagent preparation (as source of antibody).
3) Separating multiple antibodies present in plasma or elute to
aid in identification.
4) Confirmation of the presence of a weak antigen on red cells
(ABO discrepancies).
5) Confirmation of antibody specificity.

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Practical session no 11

Done by nada fallatah ,2016

 It is the process of removing antibodies from the surface of

 Antibody release (elution) can be accomplished either

1- Disrupting the linkage between antigen and antibody.

2- Changing conditions that favor dissociation of antibodies

 Lowers pH, causing antibody to dissociate.

2- Organic solvents (ether, chloroform):

3- Heat or freeze-thaw elution techniques:

 Heat (conformational change).

 Freeze-Thaw (lyses cells).

3- An albumin solution is added and the cells are heated to

5- The supernatant fluid contains the antibodies that

The eluate is tested against screening and panel cells to

Cord Blood Sample Positive for DAT.

5- Method of heat elution procedure:

complete filling of tube with saline.

6- Add 20 drops of 6% bovine albumin to the washed packed

red blood cells.

7- Place the tubes at 56°C for 10 minutes, agitating the tube

periodically during this time.

2- Lack of agglutination of test cells by the eluate indicates

 Adsorption is removal of antibody from a plasma/serum by

 Adsorption procedures can be used to investigate underlying

 ZZAP or chloroquine diphosphate can be used to dissociate

Anti-e Anti-e Anti-S

Red cells of panel for identification that carry the antigen

2- If no reactivity, that means antibody has been completely

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