2. •Antigen antibody reaction , in which a particulate
antigen combines with its antibody in the presence of
electrolytes at an optimum pH and temperature
resulting in visible clumping of particles.
•Types of Agglutination:-
•Slide agglutination test
•Tube agglutination test
•The Antiglobulin test
•Heterophile agglutination
•Passive agglutination test
Agglutination
3. The AntiglobulinTest
•Antiglobulin serum (Coombs’Serum) was
discovered by Coombs etal in 1945.
•The antiglobulin test can be used to detect red
cells sensitized with IgG alloantibodies, IgG
autoantibodies or complement components.
•Sensitization of red cells can occur in vivo or vitro.
4. Principle of AntiglobulinTest
•The incomplete antibodies (IgG) attach to red cell
membrane by the Fab (antigen binding fragment) portion
of the immunoglobulin molecule (IgG).
•The IgG molecules attached to the red cells are unable to
bridge the gap between sensitized red cells which are
separated from each other by the negative charge on their
surface and the sensitized red cells do not agglutinate.
6. What is Coombs’ Serum
•Serum from a rabbit or other animal previously
immunized with purified human globulin to prepare
antibodies directed against IgG and complement,
used in the direct and indirect Coombs' tests. Also
called antihuman globulin.
10. Indirect Coombs test (Indirect
Antiglobulin test):
The use of AHG serum to detect sensitization of red cells in
vitro is a two stage technique known as indirect
antiglobulin test (IAT)
•This test is performed to detect presence of Rh-
antibodies or other antibodies in patients serum in case of
the following:
1.To check whether an Rh-negative women (married to
Rh-positive husband) has developed Anti Rh-antibodies
2. Anti D may be produced in the blood of any Rh-negative
person by exposure to D antigen by-
•Transfusion of Rh positive blood
•Pregnancy, if infant is Rh positive (if father is Rh-positive)
11. Direct Coombs test / antiglobulin test:
The sensitization of red cells in vivo is detected by one
stage technique the direct antiglobulin test (DAT).
•Performed to detect anti-D antibody or other antibodies
attached to the red cell surface within the blood stream.
•Occurs in the following circumstances:
•When there is a Rh-positive baby in the womb of a
sensitized Rh-negative women; the antibodies produced
in the mothers serum cross the placenta and after entering
the baby's blood stream, these antibodies will attach to
the baby's Rh-positive red blood cells. These coated (or
sensitized) cells are clumped and removed from the
circulation, causing hemolytic anemia (Hemolytic Disease
of the Newborn: Erythroblastosis Fetalis).
12. When the baby is born, the baby's blood is collected (or
cord blood is collected from umbilical cord) and tested
by the anti globulin Coombs test (direct) to detect anti
D antibodies coated on red blood cells.
This mechanism could be autoimmunity, alloimmunity
or a drug-induced immune-mediated mechanism.
13. CoombsTest in Blood Banks
•The test is only rarely used to diagnose a medical
condition, but is essential for use by laboratories
such as blood banks. Blood banks use the indirect
Coombs' test to determine whether there is likely to
be an adverse reaction to blood to be transfused.
14. Hemolytic disease of the
fetus and newborn
caused by anti-D and anti-S
alloantibodies:
Case Report
15. A full-term, Chinese baby boy was born to a
30-year-oldwoman at 38 weeks of
gestation.The baby weighed 2.8 kg
The baby was noted to have mild jaundice
with normal vital signs on day one of life;
there was no evidence to suggest other
causes of neonatal jaundice such as
intrauterine infections and his glucose-6-
phosphate dehydrogenase screen was
normal. Laboratory investigations showed
that his total bilirubin was 198 μmol/L and
hemoglobin was 19 g/dL.The baby’s blood
group was A RhD positive
16. The result of a DCT was positive and red cell
elution studies of the baby’s blood identified
the presence of anti-D and anti-S antibodies.
The mother was para 3+1. Her first pregnancy
was aborted five years ago and unfortunately
no investigation was performed to find out the
cause of abortion. Subsequent pregnancies
were uneventful with no history of HDFN in
the last three years. She denied any previous
history of blood transfusion.
Her transfusion record at our center showed
that the mother developed anti-S antibodies
during her second pregnancy three years ago
17. An antenatal antibody screening test performed at
22 weeks identified only allo-anti-S; no anti-D was
detected. She was given RhD Ig prophylaxis at 28
weeks of pregnancy.
Her other laboratory investigation results showed
that she was grouped as A RhD negative with red cell
phenotype ccdee (rr), and homozygous ss. At
postpartum, the result of her DCT was negative, but
the antibody screening
test performed using the indirect Coombs’ test
method and antibody investigations showed the
presence of anti-D and anti-S, and the anti-D titer
was 1:32 (0.25IU/mL).
18. In view of the presence of allo-anti-D and allo-
anti-S in the postpartum maternal blood,
supported by the presence of similar
alloantibodies in the baby’s red cell eluates
and clinical presentation of hemolytic anemia,
a diagnosis of hemolytic disease of the fetus
and newborn secondary to anti-D and anti-S
was made.
The baby was immediately started on a course
of conventional phototherapy with a single
tungsten halogen bulb. His serum bilirubin
levels subsequently reduced to normal levels
over a few days. On the sixth day of life, the
baby was discharged well with
nocomplications.
19. Discussion
This case illustrates an uncommon case of
HDFN caused by anti-D and anti-S antibodies,
which were identified from the red cell eluate
of the baby as well as the mother’s serum
post-natally. In this case, there was no anti-D
detected at 22 weeks of gestation. However,
at postpartum,when the newborn developed
jaundice an investigation
for HDFN demonstrated that there were both
anti-D and anti-S.The possible explanation for
the anti-D at postpartum could be due to (a)
RhD Ig prophylaxis given at the early third
trimester of pregnancy.
20. The maternal serum sample was taken 40
minutes after RhD Ig immunization and
showed an anti-D titer of 1:8, which
subsequently peaked at a titer of 1:32 at 24
hours and remained at that levelfor about two
weeks and then leveled off at 1:16 fromweek
three through to week nine. At term, about
37weeks of gestation, the maternal antibody
screens reverted to normal
The high anti-D titer of 1:32 in our case study is
most probably due to RhD alloimmunization
from exposure of the mother to RhD positive
fetal red blood cells later in
21. Anti-S antibody is an IgG antibody
developed following red cell
alloimmunization. It is reactive at 37°C and
best detected by the indirect antiglobulin
test. A literature search revealed that anti-S
is a rare cause of HDFN and usually
presents as a mild form of jaundice .
However, there are a few reports of severe
and fatal HDFN due to this antibody
