Preparation of Coomb’s Control Cells

1. Dilute Eryclone anti-D (IgG)/Erybank anti-D (polyclonal) reagent 1:50 in isotonic saline.

2. Prepare a 5% suspension of group ‘O’ RhoD positive

cells in isotonic saline.

3. Mix equal volumes of diluted anti-D reagent (as in 1

above) and 5% suspension of ‘O’ RhoD positive cells

(as in 2 above) and incubate at 37°C for 15 minutes.

4. Decant and wash thoroughly with isotonic saline at

least thrice.

5. Resuspend in isotonic saline to make a 5% suspension

of coombs control cells.

Additional Material Required

For direct antiglobulin test: Test tubes (10 × 75 mm), Pasteur

pipettes, centrifuge, isotonic saline, coomb’s control cells,

optical aid.

For indirect antiglobulin test and compatibility test: Test

tubes (10 × 75 mm), Pasteur pipettes, Erybank bovine

serum albumin, centrifuge, incubator (37°C), isotonic

saline, coomb’s control cells, optical aid.

Procedure

Bring reagent to room temperature before testing.

Blood Banking (Immunohematology) 357

Direct Antiglobulin Test

1. Prepare a 5% suspension of the red cells to be tested

in isotonic saline.

2. Pipette one drop of the cell suspension into a test tube.

3. Fill the tube with fresh isotonic saline and centrifuge

for 30 seconds at 3400 rpm (1000 g).

4. Decant and repeat this washing at least thrice.

5. Add two drops of Eryclone anti human globulin

reagent and mix well.

6. Centrifuge for one minute at 1000 rpm (125 g) or for

20 seconds at 3400 rpm (1000 g).

7. Very gently, resuspend the cell button observing for

agglutination macroscopically.

8. To all negative antiglobulin tests add one drop of

Coomb’s control cells and observe for agglutination.

Indirect Antiglobulin Test

Major cross-match procedure.

Initial Phase

1. Label two test tubes as A (for albumin) and B (for

saline), depending upon the number of donors to be

cross-matched, as many pairs of such labeled tubes

would be required.

2. Prepare a 5% suspension of the red cells to be tested

in isotonic saline.

3. Pipette two drops of recipient serum in both the

labeled test tubes.

4. Pipette one drop of donor red cells in both the labeled

test tubes and mix well.

5. Only to the albumin tube (A), add two drops of

Erybank bovine serum albumin reagent and mix well.

6. Centrifuge both the tubes for one minute at 1000 rpm

(125 g) or for 20 seconds at 3400 rpm (1000 g).

7. First observe for hemolysis. Resuspend the cell button

and observe for agglutination macroscopically.

8. Proceed to incubation phase.

Incubation Phase

1. Incubate the saline tube at room temperature and the

albumin tube at 37°C for 15 minutes.

2. First observe for hemolysis. Resuspend the cell button

and observe for agglutination macroscopically.

3. Proceed to the antiglobulin phase.

Antiglobulin Phase

1. Only the albumin tubes (A) are tested in the antiglobulin phase.

2. Wash the mixture of red blood cells and serum

thoroughly with isotonic saline for minimum of three

times. Decant completely after the last wash.

3. Place two drops of Eryclone anti-human globulin

reagent into the test tubes containing the sedimented

cells and mix well.

4. Centrifuge for one minute at 1000 rpm (125 g) or for

20 seconds at 3400 rpm (1000 g).

5. Very gently, resuspend the cell button and observe for

agglutination macroscopically.

Interpretation of Results

Direct Antiglobulin Phase

Agglutination of the red blood cells is a positive test result

and indicates the presence of human IgG or components

of complement on the red blood cells.