Download Coomb`s test `` Anti Human Globulin Test`` AHG

Coomb's test '' Anti Human Globulin Test'' AHG.
METHOD OF (DAT)
• The patient's red blood cells (RBCs) are washed (removing the
patient's own serum) with saline for 3-4 times.
• Prepare 5% suspension of the washed blood.
• Add 2 volumes of AHG serum to 1 volume of the suspended cells.
• Mix, centrifuge and read for agglutination.
• If immunoglobulin or complement factors have been fixed on to the
RBC surface in-vivo, the antihuman globulin will agglutinate the
RBCs and the direct Coombs test will be positive. (A visual
agglutination).
• If still negative, IgG sensitized cells are added.
The IAT is a two-stage test.
First stage
1- Washed O red blood cells (1drop) are incubated with test serum ( 3
drops).
2- Incubate for 1 hour at 37oC (RBC sensitazation).
3- Wash the cells three times.
4- If the serum contains antibodies to antigens on the RBC surface, the
antibodies will bind onto the surface of the RBCs.
Second stage
5- Add two drops of antihuman globulin mix.
6- Centrifuge for 15-20 sec at 1000 g.
7- Read for agglutination.
8- Record reslt.
9- If still negative, leave it at RT for 5 min, re centrifuge and examine.
10If still test negative, add 1 drop of IgG- coated cells.
11- Repeat step 6 and 7.
12- A positive reaction indicates that the negative result in step 8 is
valid, but if the control IgG-coated cells fail to agglutinate, then the
test should be repeated.
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