EXPERIMENT NO.

14
DIRECT ANTIGLOBULIN TEST (DIRECT COOMBS’ TEST)

1. Label a 12x75 mm test tube.

2. Place 2 drops of patient’s red cell suspension (freshly prepared).
3. Centrifuge, decant as much supernatant as possible.
Note: Excess diluents may dilute the AHG reagent.
4. Add a drop of AHG reagent.
5. Mix gently. Centrifuge 2500 rpm for 30 seconds.
6. Re-suspend the cells using the tilt and wiggle technique.
7. Observe for agglutination. Place a small amount of the mixture on the slide and observe
under the microscope. Grade and record the reaction.
8. If using polyspecific AHG or antiC3d, incubate non-reactive tests at room temperature
for 5 minutes, then centrifuge, and read again.
9. Confirm the validity of negative tests by adding IgG-coated red cells to tests containing
anti-IgG.
10. Examine the cells for agglutination and record the reaction.

Interpretation of Result:
1. Clumping of red cells: Positive DAT
 Indicates in-vivo coated red cells
 N.B. IgG red cells usually give immediate reactions, whereas complement coating
may be more easily demonstrable after incubation
2. Free Cells: Negative DAT
3. The IgG-coated control cells are expected to be agglutinated. If they are not
agglutinated, negative DAT result is considered INVALID and must repeat test.

EXPERIMENT NO.15
INDIRECT ANTIGLOBULIN TEST (INDIRECT COOMBS’ TEST)
1. Label the tube with patient’s name.
NOTE: Partner groups may be assigned for sharing serum and red cells during the
testing.
2. Place 2 drops of patient’s serum in a labeled 12x75 mm or 8x75 mm test tube.
3. Add 1 drop of (another group’s) red cell suspension.
4. Incubate the tube for 30 minutes at 37 C.
5. Wash the set up with NSS 3 times to remove proteins and un-reacted antibodies.
N.B. Place NSS ¾ full of the best. Mix and centrifuge. Decant the supernatant carefully.

6. After the last wash, remove as much as possible the washing fluid, but without
disturbing the cell button.
7. Add 2 drops of AHG reagent.
8. Centrifuge for 30 seconds.
 9. Re-suspend the cells gently employing the tilt and wiggle technique.
10. Observe the reaction and grade the strength of agglutination.
11. Transfer a small amount of the mixture onto the slide. Observe the reaction under the
microscope.

Interpretation of Result:
Agglutination of Red Cells: Positive IAT (meaning IgG antibodies are bound to the red cell
antigens.
No Agglutination of Red Cells: Negative IAT