Direct Antiglobulin test

1. Antiglobulin Test
(Coomb’s Test)

2. The antiglobulin test
• The antiglobulin test was first introduced into
clinical medicine in 1945 by R.R. Coombs
• He showed that it could be used to detect non-
agglutinating red cell antibodies or sensitized
red cells
• Most non-agglutinating (incomplete) antibodies
are IgG,
• These antibodies do not spontaneously cause
agglutination due to a strong electronegative
charge on the red cell surface that prevents the
cells from coming into close proximity.
• The antiglobulin reagent is able to bridge these
negative forces.

3. Antihuman Globulin
Definition:
• Antihuman: antibodies against human antigens
• Globulin: all antibody molecules are globulins
Therefore: Antihuman Globulin is antibody directed
against the Fc portion of human antibodies
and/or complement components.

4. AHG Technique: What is the
importance?
• Some very clinically significant unexpected
antibodies (eg. Kidd, Duffy) attach to red cell
but do NOT cause agglutination at immediate
spin or 37o phase.
• Yet, these antibodies are capable of causing
severe hemolytic transfusion reactions or
hemolytic disease of the newborn.

5. ANTIGLOBULIN TEST
• Detection of non-agglutinating Abs,
especially IgG or complement components
(C3) attached to RBCs in vivo or in vitro.
– Direct antiglobulin test (DAT) - detection of
sensitization of RBC s (coated with Abs
and/or complement components) in vivo
– Indirect antiglobulin test (IAT) - detection of
sensitization of RBCs (coated with Abs and/or
complement components) in vitro

6. Antiglobulin Test
• Principle - Antihuman globulins (AHG)
from immunized animals bind to human
globulins either free in serum or
attached to RBCs
– Pentameric IgM Abs are so large that,
when bound to RBC Ags, the RBCs
agglutinate (usually at RT)
– IgG Abs usually need a little help, a bridge
molecule, to agglutinate RBCs
– AHG acts as a bridge molecule

7. Reagent Preparation
• Polyclonal or monoclonal sources
–Polyclonal - Animals immunized
with human globulins (IgG &
complement (C3); bled for antisera
to obtain high titered
–Monoclonal - Hybridoma cells

8. Reagent Preparation
• Polyspecific AHG
– Abs to human IgG, and
– Abs to human C3d (C3b breaks down to C3c and
C3d)
– Advantage is that polyspecific AHG may detect
complement-dependent Abs on RBCs
• Monospecific AHG
– Abs to human IgG only or human C3d only

9. Antiglobulin Test

10. Direct Antiglobulin Test
• Detects in vivo sensitization of RBCs
• The DAT is used to detect IgG or C3
bound to the surface of the red cell.
• In patients with hemolysis, the DAT is
useful in determining whether there is an
– Immune etiology
– OR Non-immune etiology of hemolysis

11. Direct Antiglobulin Test

12. Non-immune causes of hemolysis
• Mechanical hemolysis such as those due
to artificial valves or burns,
• Hemoglobinopathies (sickle cell,
thalassemia),
• Red cell enzyme deficiencies (G6PDP,
pyruvate kinase),
• and red cell membrane defects (hereditary
spherocytosis, PNH)
Have a negative DAT

13. DAT Uses
 Immune causes of hemolysis
 Autoimmune hemolytic anemias,
 Drug induced hemolysis,
 Delayed or acute hemolytic transfusion
 Hemolytic Disease of the new born
Have a positive DAT

14. Indirect Antiglobulin Test (IAT)
• Detection of in vitro sensitization of RBCs
• Most clinically significant alloantibodies are IgG
antibodies that react best at 37oC and are
formed as a result of previous exposure via
transfusion or pregnancy.
• Examples include antibodies to Rh, Kell, Kidd,
and Duffy red cell antigens.
– Same as DAT, except:
– Step 1 entails incubating RBCs (reagent or unknown)
with antisera (reagent or unknown) allowing time for
in vitro attachment of Abs to RBCs

15. Indirect Antiglobulin Test

16. IAT Uses
• When the unknown is sera:
– Ab Screening: The patient’s serum is incubated
with red cells of known phenotype to detect Ab in
patient’s serum against a specific red cell Ag
– Phenotyping: Ab of known specificity is incubated
with patient’s RBC to identify specific blood group
Ag on the cells
– Crossmatch: The patient’s serum is incubated with
donor red cells to detect Abs that might reduce the
survival of transfused red cells

18. Modifications of IAT
• Modifications can be done to increase
sensitivity & to decrease time to perform
IAT
– Modification of suspending media
– Modification of RBCs
• Each modification has advantages and
disadvantages

19. Modification of suspending medium
suspending
medium
Mechanism of
action Advantages Disadvantages
LISS
Facilitates
sensitization but
diminishes
agglutination
Short incubation
time
expensive
Albumin
Facilitates
sensitization
and
agglutination
Short incubation
time, enhances
certain clinically
significant Abs
Enhances the
reaction
caused by cold
autoantibodies

20. Importance of Anti-complement in
antiglobulin reagents
• Anticomplement increases detection
sensitivity for certain Abs (Anti-Kidd)
• Approx. 200 molecules of cell bound IgG
required for +ve antiglobulin test
• Ab sensitization below this level will not be
detected by anti-IgG alone
• If antiglobulin reagent contains both anti-
IgG & anti-C3b will increase probability of
detection of sensitization

21. False Positive Reactions
False Positive Mechanism
Cold Autoantibody
“in vitro” complement fixation &
auto-agglutination
Technical Dirty tubes
Anti-species Abs
Heterologous Ab in the
antiglobulin reagent
Polyagglutinable
red cells
Presence of anti-T or anti-Tn in
the antiglobulin reagent

22. False Negative
Technical
problems
Failure to add reagent
Neutralization of the antiglobulin
reagent due to insufficient washing of
the test cells
Contamination or neutralization of
antiglobulin reagent
False –ve test can be detected by addition of IgG sensitized cells to –ve
antiglobulin tests. These cells should be agglutinated by the anti-IgG