Test tube method is more reliable than slide test, but takes longer time and more equipment. For cell grouping, patient’s saline-washed red cells are mixed with known antiserum in a test tube; the mixture is incubated at room temperature, and centrifuged. For serum grouping, patient’s serum is mixed with reagent red cells of known group (available commercially or prepared in the laboratory), incubated at room temperature, and centrifuged (See Table). Following centrifugation, a red cell button (sediment) will be seen at the bottom of the tube. Cell button is dislodged by gently tapping the base of the tube and examined for agglutination.
Positive (+) Test
Clumps of red cells suspended in a clear fluid. Agglutination in tube test is graded from 1+ to 4+ and read macroscopically (See Figure). 
Grading of ABO tube test
Grading of ABO tube test. Negative: Uniform suspension of red cells; Grade 1 (1+): Many small clumps of red cells (fine granular appearance); Grade 2 (2+): Many large clumps with many free red cells; Grade 3 (3+): Three or four individual clumps with few free red cells; and Grade 4 (4+): One solid clump of red cells with no free red cells
Negative (–) Test
Uniform suspension of red cells.

Separate tubes of auto-control, positive control, and negative control should always be setup along with the test sample tube. Auto-control tube consists of mixture of patient’s red cells and patient’s own serum. This is required to rule out false-positive result due to auto antibodies in patient’s serum causing auto agglutination of patient’s own red cells. Auto-control test is particularly essential when ABO grouping is being done only by forward method and blood group is typed as AB. If there are auto antibodies in recipient’s serum, ABO grouping, Rh typing, antibody screening, and cross matching all will show positive result.
In two positive control tubes, anti-A serum is mixed with group. A red cells and anti-B is mixed with group B red cells respectively. In two negative control tubes, anti-A serum is mixed with group B red cells and anti-B serum is mixed with group. A red cells respectively. These controls are necessary to confirm that reagents are working properly.
Interpretation of forward (cell) and reverse (serum) grouping
Interpretation of forward cell and reverse serum grouping
Why test tube method of blood grouping is more reliable than slide method?
Test tube method of blood grouping is more reliable than slide method. This is because centrifugation enhances the reaction by bringing antigen and antibodies closer together and allows detection of weaker antigen antibody reactions; in addition drying is avoided and smaller amounts of reagent are required.
If forward grouping, reverse grouping, and autocontrol tests are all positive, then these results are probably indicative of a cold-reactive autoantibody. Before performing forward typing, red cells should be washed with normal saline to elute the antibody. Before performing reverse grouping, autoantibody should be adsorbed by washed cells till autocontrol is negative.


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