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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. 7