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Antiglobulin (Coombs) Test
Introduction
• Antiglobulin serum (Coombs’ Serum) was
discovered by Coombs in 1945.
• The antiglobulin test can be used to detect red
cells sensitized (coated) with IgG
alloantibodies, IgG autoantibodies or
complement components.
• The process of RBCs sensitization can occur in
vivo or vitro.
PRINCIPLE of the test
• Normal human red blood cells, in presence of antibody
directed towards the antigen they possess, may fail to
agglutinate when centrifuged and become sensitized. This
may be due to the particular nature of the antigen and
antibody involved.
• Sensitization of RBC’s may be with IgG or complement.
• In order for agglutination to occur an additional of anti-
antibody or anti-complements, at which the Fab portion
of AHG reacts with the Fc portion of the IgG antibody, or
with the C3b or C3d component of complement
alternatively.
• AHG will form a bridge between the
antibodies or complement coating the red
cells, causing agglutination.
AHG
• Can be:
1. Polyspecific (broad spectrum) reagent: this
reagent contains both anti-Ig and anti-
complement (blended of Anti-IgG and Anti-
C3b, -C3d)
2. Monospecific reagent: directed against
specific immunoglobulin or component of
complement.
Antiglobulin (coombs) Test
Two types:
1. Direct antiglobulin test (DAT)
2. Indirect antiglobulin Test (IAT)
Direct antiglobulin (coombs) test {DAT}
• DAT detects antibodies coating the surface of
erythrocytes (in-vivo sensitization).
• Erythrocytes are washed and with saline to
removed unbound antibodies, and then anti-
human globulin (AHG) reagent is added.
• The Fab portion of AHG attaches to the Fc
portion of the bounded antibodies or to
complement component resulting in visually
detected agglutination.
Direct antiglobulin test (DAT)
AHG
In-vivo Sensitized
RBCs
Direct antiglobulin (coombs) test {DAT}
• Applications:
1. Autoimmune hemolytic anemia

2. Drug induced hemolytic anemia
3. Hemolytic disease of newborn (HDN)
4. Suspected hemolytic transfusion reaction (HTR)
DAT procedure
1. Add 1 drop of 2-5% suspension of washed
RBCs.
2. Add 2 drops of polyspecific AHG reagent.
3. Mix well and immediately centrifuge at 1000
rpm for 15 seconds.
4. Check for agglutination.
5. To confirm negative results, use CC (check
cells, control cells or coombs cells)
Indirect antiglobulin (coombs) test {IAT}
• IAT identified antibodies in the tested serum.

• Serum will be incubated with erythrocytes to
allow in-vitro sensitization of the RBCs.
• The RBCs are then washed with saline to
remove unbounded antibodies.
• Finally, the AHG is added.
Indirect antiglobulin (coombs) test {IAT}
• Applications:
1. To detect/ identify the presence of antibodies in the

recipient's serum (Panel Test).
2. To test the donor red cell antigens.
3. To confirm the absence of antigen-antibody
reaction, where recipient's serum is tested against
donor blood cells ( compatibility/cross match).
IAT procedure
• The tested serum is firstly should incubated with
normal properly washed RBCs. Immunoglobulins
or complements (if present on the serum) will
allow sensitization of these red cells.
• If sensitization occurs, a positive result for
agglutination will obtained after the addition of
AHG.
• Also negative results should be confirmed by
using CC.
Cross matching
Cross-match
Major and Minor cross-match tests

• Major cross-match test, consisting of mixing the patient’s
(recipient) serum with donor RBCs.
• Minor cross-match test, consisting of mixing the donor’s
plasma with patient’s RBCs
• Cross-match methods can be categorized by
the test phase in which the procedure ends.
• Three phases:
1. Immediate spin / room temperature
2. Incubation phase (37ºC incubation)
3. Coombs phase / indirect antiglobulin test
Immediate spin (IS)
• In IS (the patient’s serum with donor cell are

centrifuge immediately) absence of hemolysis
or agglutination indicates compatibility.
• The purpose of the immediate spin is to
detect "cold" antibodies, usually of the IgM
class (ABO or other cold reactive antibodies).
Incubation phase
• Two drops of bovine albumin or LISS (low ionic
strength solution) should be added then
incubate at 37ºC for 15-30 minutes.
• Centrifuge and check for agglutination.
WHY????
• Both work as enhancement media (or
potentiator) to speed up the reaction (see
slides later)
Coombs (AHG) phase
Antiglobulin Cross-match
• The procedure begin in the same manner as the IS
cross-match, continues to 37°C incubation and
finishes with AHG test.
NOTE:
• When negative, you should confirm the validity of test using Abs
coated RBC (CC, check cells, control cells or coombs cells).
Result interpretation:
• If all tubes have remained negative
throughout the cross match procedure (with
positive result in control cells), then the unit is
interpreted as “compatible”
• When incompatibility found, the nature of
antibodies that recipient has should be
identified.
• If CC result is negative ?????
Cross-match
No agglutination ~ compatible
Agglutination ~ incompatible
Donor RBCs Patient serum

(washed and
suspension)
column (gel) agglutination
• Column (gel) agglutination are beginning to
gain widespread use in clinical laboratories.
Instead of a test tube, the reactions are
carried out in micro-tubes, which contain a
dextran-acrylamide gel.
Gel testing.
• Agglutination is graded on a scale from 0 to 4+.
• A: 4+ reaction
• B: 3+ reaction
• C: 2+ reaction
• D: 1+ reaction
• E: 0 no agglutination and red blood cells pass all the way to the bottom.
gel agglutination
POTENTIATORS
• Some antibodies will not react in a saline
environment.
• Potentiators are reagents that adjust the test
environment.
 Reduce the zeta potential
 Promote agglutination
Zeta Potential
• RBCs have a negative surface charge, and
attract cations such as Na+.
• The electrical potential between the red cell
surface and the outer ionic cloud surrounding
it is the zeta potential.
Zeta Potential
By reducing the ionic
cloud, the potentiator
allows red cells to come
closer together.
 22% Albumin -----------

(serological albumin)
 LISS: Low Ionic Strength
Solution, made of NaCl,
glycine and albumin

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Antiglobulin (Coombs) Test

1. Autoimmune hemolytic anemia

• IAT identified antibodies in the tested serum.

1. To detect/ identify the presence of antibodies in the

Major and Minor cross-match tests

• In IS (the patient’s serum with donor cell are

Donor RBCs Patient serum

 22% Albumin -----------

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