Antibody Titration

PROCEDURE
Prepare 5% RCS of the known

antigen specific for the antibody
whose titer is being determined
(Known A and Known B cells).
Prepare serum sample from the

coagulated blood of the patient.
Determine the antibody to be

titrated. Antibody being measured
usually corresponds with the
antigen present in the fathers
blood.
Prepare a set of 12 appropriatelysized test tubes, and carry out

serial dilutions as follows:
Tube #
Volume of NSS in
Volume of Patients
ml
serum in ml
Dilution
0.1
0.1
0.1
1:2
0.1
0.1 from tube 2
1:4
0.1
0.1 from tube 3
1:8
0.1
0.1 from tube 4
1:16
0.1
0.1 from tube 5
1:32
0.1
0.1 from tube 6
1:64
0.1
0.1 from tube 7
1:128
0.1
0.1 from tube 8
1:256
10
0.1
0.1 from tube 9
1:512
11
0.1
0.1 from tube 10
1:1024
12
0.1
0.1 from tube 11
1:2048
Add appropriate cells to each

tube.
Spin down. Agitate tubes gently

then evaluate for the presence of
agglutination.
Record
your
observation. Set aside tubes with
agglutination.
Add 2 drops of 22% Bovine Serum

Albumin to each of the tubes that
have no visible agglutination.
Incubate the tubes treated with

BSA at 37 degrees Celsius for one
hour.
Wash the incubated tubes with

cold NSS 2-3 times. Completely
decant the saline after washing.
10
Add 2 drops of AHG reagent to

each tube and re-suspend the
cells by gentle agitation.
11
Read the tubes macroscopically

and microscopically. Interpret the
results. If the titer is >256, the titer
for the particular antibody is
elevated.
Questions for
Research
What is the principle of the test?

Twofold serial dilutions of serum
containing an antibody are prepared
and tested against a suspension of
RBCs that possesses the target antigen.
The titer level is the reciprocal of the
greatest dilution in which agglutination
of 1+ or greater is observed.
What is the purpose of the cold

NSS in the procedure?
Cold NSS is used to wash the red cells
prior to the addition of the AHG
reagent in order to detect low affinity
IgG molecules that is lost if washing is
done with NSS at room temperature.
Why is there a need to wash the

red cells before adding the AHG
reagent?
Washing the red cells prior to the
addition of the AHG reagent will
remove unbound antibodies to allow
the AHG reagent to aid in the
agglutination of incomplete antibodies.
Post-Lab
Conference
What are common sources

of error for this test?
List down conditions and/

or situations where Group
O
blood
may
be
transfused to a non-Group
O recipient. Indicate the
antibody titer which may
be considered safe.
Group O blood can be used as an

Alternative if the patient/ recipient is
Group A, B, AB blood.
For patients of Group A or B, the
second choice should be Group O,
provided that it is plasma depleted
and/or does not contain high-titer
hemagglutinins.
Group O Rh-negative red blood cells

may be used while awaiting results of
the group. If the Group O Rh-negative
supply becomes compromised, the
patient should be maintained with
Group O positive until such time as the
patients group can be determined.
In extreme emergencies, when there

is no time to obtain and test for pretransfusion sample, Group Rh-negative
packed cells can be used.
In an emergency, Rh-positive blood
may be transfused to non-immunized
Rh-negative men and older women, if
Rh-negative blood is not available and
if blood transfusion will be potentially
lifesaving.
ANTIBODY TITERS which

are considered SAFE:
When the Antibody Titer is greater than
64 (>10 IU/ml anti-D), no part of the
donation is issued for clinical use nor for
fractionation of the plasma for blood
products.

When the Antibody Titer is less than 64
(or <10IU/ml anti-D), the red cells may be
released for transfusion after separating
most of the plasma from the donation and
replacing it with an optimal additive
medium; the plasma may be used for
fractionation.

When the Antibody Titer is less than 8 (<5
IU/ml anti-D), the donation may be used
either as whole or plasma-reduced blood.
Thanks!
ANY QUESTIONS?
References
AABB Technical Manual, 15th Edition, pages
761 763
Care of the Critically Ill Patient edited by
Jack Tinker, Warren Zapol, page 709
Textbook of Adult Emergency Medicine by
Peter Cameron, pages 566-567
Modern Blood Banking and Transfusion
Practices, Denise M. Harmening, pages 220,
232, 460
Modern Transfusion Medicine, Derwood
Pamphilon, page 45
Immunology and Serology in Laboratory
Medicine, Mary Louise Turgeon, page 24

Antibody Titration

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Antibody Titration

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What is the principle of the test?

What is the principle of the test?

What is the purpose of the cold NSS in the procedure?

What is the purpose of the cold NSS in the procedure?

PROCEDURE

Prepare 5% RCS of the known

Prepare serum sample from the

Determine the antibody to be

Prepare a set of 12 appropriatelysized test tubes, and carry out

0.1 from tube 2

0.1 from tube 3

0.1 from tube 4

0.1 from tube 5

0.1 from tube 6

0.1 from tube 7

0.1 from tube 8

0.1 from tube 9

0.1 from tube 10

0.1 from tube 11

Add appropriate cells to each

Spin down. Agitate tubes gently

Add 2 drops of 22% Bovine Serum

Incubate the tubes treated with

Wash the incubated tubes with

Add 2 drops of AHG reagent to

Read the tubes macroscopically

What is the principle of the test?

What is the purpose of the cold

Why is there a need to wash the

What are common sources

List down conditions and/

Group O blood can be used as an

Group O Rh-negative red blood cells

In extreme emergencies, when there

ANTIBODY TITERS which

ANTIBODY TITERS which

ANTIBODY TITERS which

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