Practical
Blood Bank
Antibody Titration

Titration is a semi quantitative method used to
determine the concentration of antibody in a
serum sample or to compare the strength of
antigen expression on different red cell samples.
The usual applications of titration studies are:
1. Estimating antibody activity in alloimmunized
pregnant women to determine whether and when
to perform more complex invasive investigation of
the fetal condition.
2. characterizing antibodies as high-titer, low-avidity
3. Observing the effect of sulfhydryl reagents
on antibody behavior, to determine
immunoglobulin class (IgG or IgM).
Titration studies specifically to assist in
monitoring clinically significant antibodies in
the pregnant woman.


Speed and strength of agglutination is
termed as avidity.
The test is done by mixing two drop of antiserum with one drop of 40-50% cell
suspension on a slide or tile and rocking
gently at room temperature (RT). The time for
a clearly visible reaction (+1) and then for
strong (+4) reaction to occur are recorded
with the help of stop watch.

Serum for titration (containing potentially significant
unexpected antibodies to red cell antigens, 1 mL). If
possible, test the current sample in parallel with the
most recent previously submitted (preceding)
sample from the current pregnancy.
1. Antihuman IgG: need not be heavy chainspecific.
2. Isotonic saline.
3. Volumetric pipettes, or equivalent: 0.1- to 0.5-mL
delivery, with disposable tips.
4. Red cells: group O reagent red cells, 2%
suspension. (See note 1 regarding the selection
of red cells for testing.) Avoid using Bg+ red cells
because they may result in falsely high values,
especially with sera from multiparous women.
5. IgG-coated red cells.
1. Using 0.5-mL volumes, prepare serial twofold dilutions
of serum in saline. The initial tube should contain
undiluted serum and the doubling dilution range
should be from 1 in 2 to 1 in 2048 (total of 12 tubes).
2. Place 0.1 mL of each dilution into appropriately
labeled test tubes.
3. Add 0.1 mL of the 2% suspension of red cells to each
dilution. Alternatively, for convenience, add 1 drop
of a solution of a 3% to 4% suspension of red cells as
supplied by the reagent manufacturer, although this
method is less precise.
4. Gently agitate the contents of each tube;
incubate at 37 C for 1 hour.
5. Wash the red cells four times with saline;
completely decant the final wash supernatant.
6. To the dry red cell buttons thus obtained, add antiIgG according to the manufacturer’s directions.
7. Centrifuge as for hemagglutination tests.
8. Examine the red cells macroscopically; grade and
record the reactions.
9. Add IgG-coated red cells to all negative tests;
recentrifuge and examine the tests for
macroscopic agglutination; repeat the testing if
the tests with IgG-coated red cells are
nonreactive.

The titer is reported as the reciprocal of the highest
dilution of serum at which 1+ agglutination is
observed. A titer ≥16 (this value may vary
according to the laboratory) is considered
significant and may warrant further monitoring for
HDFN.
1. The selection of the most suitable phenotype of
red cells to use when performing titration studies
for HDFN is controversial.
◦ Some workers select red cells that have the strongest
expression of antigen, such as R2R2 for anti-D.
◦ it is important that the laboratory be consistent and use red
cells of the same phenotype for future titrations to test the
same patient’s serum.
2. Titration studies should be performed upon initial
detection of the antibody; save an appropriately
labeled aliquot of the serum (frozen at –20 C or
colder) for comparative studies with the next
submitted sample.
3. When the titer (eg, >16) and the antibody
specificity have been associated with HDFN, it is
recommended that repeat titration studies be
performed every 2 to 4 weeks, beginning at 18
weeks’ gestation; save an aliquot of the serum
(frozen at –20 C or colder) for comparative studies
with the next submitted sample.
4. Do not use enhancement techniques [albumin,
polyethylene glycol, low ionic strength saline (LISS)]
or enzyme-treated red cells because falsely
elevated titers may be obtained. Gel testing is not
recommended.
5. LISS should not be used as a diluents in titration
studies; nonspecific uptake of globulins may occur
in serum-LISS dilutions.
6. Failure to obtain the correct results may be
caused by
 incorrect technique, notably, failure to use
separate pipette tips for each dilution or
 failure to adequately mix thawed frozen serum.



If titrating anti-A, anti-B, or anti-A,B, the
serologic technique is performed by the
same method as ABO Typing
If titrating Rh, Kell, Duffy, or Kidd antibodies,
the serologic technique includes a 37oC
followed by antiglobulin testing.
Prozone phenomenon may occur so the first
tubes may have a weaker reaction than the
more diluted serum. AABB recommends
reading the most dilute tubes first and then
shake out the other tubes.
Grade
Description
+4
Single clump of agglutination with no free cells
+3
Three or four individual clumps with few free cells
+2
+1
Many fairly large clumps with many free cells
+W
2 to 3 cells sticking together per low power field, uneven
distribution Visually no agglutination
Fine granular appearance visually, but definite small
clumps (10-15 cells) per low power field
-
All cells are free
H
Hemolysis (partial or total) must be interpreted as
positive
0
No agglutination
or Hemolysis
W+ to 1+
Tiny
agglutinates
turbid
background
Small
agglutinates
turbid
background
2+
Medium-sized
Agglutinates-Clear
Background
3+
Several large agglutinates-Clear
Background
4+
One Solid Agglutinate