Coombs test

In immunological hemolytic anemias

antibodies targeting a RBC antigen bind to
the membrane. If they do not activate
complement (called incomplete
antibodies), no RBC lysis occurs.

However, antibody-coated RBCs are

captured in the spleen and destroyed.
Coombs test: to detect incomplete
antierythrocyte antibodies
Direct Antiglobulin Test (DAT): To detect
antibodies bound to RBC surface.

If cells are coated with IgG, an anti-IgG antiserum will

generate bridges among cells, thus causing red cell
agglutination.

Patient’s blood.
Obtain washed RBCs (to eliminate all plasma IgG).
Add antiglobulin antiserum.
Incubate 15 min 37°C.
Look for agglutination (visual, microscope).
 Indirect Antiglobulin Test (IAT): To detect
antierythrocyte antibodies free in serum.

Free antibodies may be present in serum if:

• Large amount of antibody, part of which
still unbound
• Antibodies against an antigen that is not
present on patient’s own RBC

Let normal RBCs absorb IgG from patient’s

serum, then do the direct test.
 Indirect Coombs test
Patient’s serum.
Washed normal RBCs, group 0 (or compatible)
Incubate 15 min 37°C
Wash incubated RBCs (to eliminate serum IgG).
Add antiglobulin antiserum.
Incubate 15 min 37°C
Look for agglutination (visual, microscope).
Rh(D)-related newborn hemolytic anemia

• Mother: Rh(D) negative

• 1st child Rh(D) positive
• Mother sensitization (usually at labor)

• 2° child Rh(D) positive

• Anti-Rh(D) antibodies cross the placenta
and cause fetal RBC lysis
Coombs test for newborn hemolytic anemia

• Newborn (2° child): is Rh(D) positive and has

circulating antibodies (which have crossed the
placenta) bound on his/her RBCs: do DAT

• Mother: is Rh(D) negative. Anti-Rh(D) antibodies

will never bind to her RBCs. Look for free
antibody in serum by the indirect test (IAT),
using normal Rh(D) red cells.