Direct Anti-Human Globulin test (DAT)

Aims:
To detect the presence of antibody and/or complement directly on an individual's
RBCs due to in vivo sensitization.
Specimen:
- Red cells suspension.
Materials:
- Pastier pipettes.
- Normal saline (0.9%).
- Glass tubes 75x12 mm.
- AHG (Polyclonal reagent i.e. Anti-IgG + Anti-C3).
- 2 % S.S.A
Procedure:
1. Take 2-3 drops of blood to be tested in a clean labeled test tube.
2. Wash the red cells 3 times (Centrifuge at 3000 rpm for 3 minute) in a large
volume of saline ( to remove free globulin molecules) . Remove all supernatant
after each wash. Completely decant the final supernatant wash.
*last wash check by 2 % S.S.A if clear go to second step(3) if present turbidity
rewash the cell and go to step (3) .
3. Add 2 drops of polyspecific AHG to washed red cells.
4. Mix, Centrifuge at 1000 rpm for 1 minute.
5. Gently shake the tube to dissolve the cell button and see for agglutination,
macroscopically if not clear read microscopically, then Record the result.

Result: Interpretation:

If give agglutination that mean a indicates an in-vivo sensitization of

positive (DAT) red cells.
If no agglutination that mean a negative No indicates an in-vivo
(DAT) sensitization of red cells.
Indirect Anti-Human Globulin test (IAT)
Aims
To demonstrate antibodies in serum sample that may cause red cells sensitization
“in vitro”.
Principle:
The antibody-containing serum is incubated with specific RBCs, followed by
washing, and then AHG serum is added to see whether RBC sensitization has
occurred.
Specimen:
- Fresh Serum.
Materials:
- Pastier pipettes.
- Normal saline (0.9%).
- Glass tubes 75x12 mm.
- Reagent cells
- AHG (Polyclonal reagent i.e. Anti-IgG + Anti-C3).
Procedure:
1. Add 2 drops of serum sample to properly labeled tubes.
2. Add 1 drop of 5% saline-suspended reagent cells and mix.
3. Incubate at 37° C for 30 to 45 minutes.
4. Wash the red cells 3 times (Centrifuge at 3000 rpm for 3 minute )in a large volume
of saline ( to remove free globulin molecules) . Remove all supernatant after each
wash. Completely decant the final supernatant wash.
*last wash check by 2 % S.S.A if clear go to second step(6) if present turbidity
rewash the cell and go to step (6) ..
6. Add 2 drops of polyspecific AHG to washed red cells.
7. Mix, Centrifuge at 1000 rpm for 1 minute.
8. Gently shake the tube to dissolve the cell button and see for agglutination,
macroscopically if not clear read microscopically, then Record the result.
Interpretation:
Result: Interpretation:

If give agglutination that mean a Indicates a presence of atypical IgG

positive (IAT) anti-body in the serum results red cells
sensitization “in vitro”.

If no agglutination that mean a negative Indicates no presence of atypical IgG

(IAT) anti-body in the serum also no red cells
sensitization “in vitro”.
 Du method
Aims
To test red cells for the presence of weak D antigen (which can't be detected with
direct Rh D typing method) using AHG globulin. The procedure is one of the
applications of in-direct anti-human globulin (IAT).
Specimen:
- 5% red cell suspension.
Materials:
- Pastier pipettes.
- Normal saline (0.9%).
- AHG (Polyclonal reagent i.e. Anti-IgG + Anti-C3).
Procedure:
1. Place 2 drops of anti-D in a clean, labeled test tube.
2. Add 1 drop of 5% saline-suspended patient cells and mix .( A tube for direct
Rh D grouping and gave negative result can be used) .
3. Incubate at 37° C for 15 to 30 minutes.
4. Wash the red cells 3 times (Centrifuge at 3000 rpm for 3 minute) in a large
volume of saline (to remove free globulin molecules). Remove all supernatant
after each wash. Completely decant the final supernatant wash.
*last wash check by 2 % S.S.A if clear go to second step(5) if present turbidity
rewash the cell and go to step (5) ..
5. Add 2 drops of polyspecific AHG to washed red cells.
6. Mix, Centrifuge at 1000 rpm for 1 minute.
7. Gently shake the tube to dissolve the cell button and see for agglutination,
macroscopically if not clear read microscopically, then Record the result.
Result: Interpretation:

If give agglutination that mean a Rh D+ve by Du method

positive reaction

If no agglutination that mean a negative Rh D-ve by Du method

reaction

Prepare by Emtenan Hassan