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4-DAT, IDAT and Du Method

This document describes procedures for three blood typing tests: 1) The Direct Antiglobulin Test (DAT) detects antibodies on red blood cells and indicates in vivo sensitization. 2) The Indirect Antiglobulin Test (IAT) detects antibodies in serum that cause in vitro sensitization of red blood cells. 3) The Du test detects weak D antigens on red blood cells using antihuman globulin, and is a type of IAT.

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Cabdalle Kurbe
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60 views

4-DAT, IDAT and Du Method

This document describes procedures for three blood typing tests: 1) The Direct Antiglobulin Test (DAT) detects antibodies on red blood cells and indicates in vivo sensitization. 2) The Indirect Antiglobulin Test (IAT) detects antibodies in serum that cause in vitro sensitization of red blood cells. 3) The Du test detects weak D antigens on red blood cells using antihuman globulin, and is a type of IAT.

Uploaded by

Cabdalle Kurbe
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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

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