Cold Agglutinins Procedure


Cells heavily coated with auto-anti-I (cold agglutinins) may agglutinate spontaneously or during centrifugation when the blood is cooled to 30oC or lower even when no other antigen-antibody reaction has taken place. Washing the cells in warm saline will elute the antibody off the cells so they can be typed.

A high concentration of auto-anti-I in the serum may agglutinate cells from all adult donors, including reagent A1 and B cells and screening cells. It must therefore be removed from the serum before the patient can be backtyped or before clinically significant alloantibodies can be identified. Since it is an autoantibody, it may be absorbed onto the patient's own cells in the cold, if the patient has not been recently transfused.

When autoadsorbing, there may be such a high concentration of anti-I in the serum that it will first be necessary to treat the red cells with a proteolytic enzyme such as papain. This modifies the red cell membrane so that greater quantities of auto anti-I may be adsorbed onto the cell.


If the cold agglutinins are not too strong, the red top tube used for the type and crossmatch will be sufficient.

If the cold agglutinins are strong, you will need one 10 ml clotted (red top) tube and two 7-ml EDTA (lavender top) tubes to autoadsorb.


  • Plastic bags
  • Wash bottle with physiologic saline warmed to 37C
  • Dispo Pipets
  • 12 x 75 mm tubes
  • Serofuge
  • Lighted agglutination viewer
  • Equipment listed above
  • Larger tube - approximately 15 x 75 mm
  • 37oC waterbath
  • Refrigerator
  • (Freeze-dried papain)


  1. Warm a wash bottle containing physiologic saline to 37C by placing it in a plastic bag and putting it in the 37C waterbath for 30 minutes. Take care that water does not enter the saline bottle.
  2. Wash 5 drops of the patient's cells at least twice with the warm saline. Return the saline bottle to the waterbath each time so that it stays warm.
  3. Make a 3% suspension of the washed cells, using warm saline.
  4. Test for antibody removal:
  • Transfer 2 drops of the 3% suspension to another 12 x 75 mm tube and centrifuge 45 seconds at high speed in the serofuge.
  • Gently resuspend and examine for agglutination. If there is no agglutination in this step, the cells are ready for typing.
  • If there is agglutination in step b, the cells must be washed further. Repeat steps 3 and 4a-b until the cells no longer agglutinate in the saline.


  1. Place the red top tube in the refrigerator and the lavender top tubes in the 37oC waterbath, wrapped in a plastic bag. Allow at least 30 minutes for the tubes to reach the desired temperature.
  2. Centrifuge the lavender top tubes two minutes; remove and discard the plasma, or save for further testing if desired. Leave the red top tube in the refrigerator for now.
  3. Wash the tubes at least twice with the warm saline. Replace the stopper and invert several times with each addition of saline to ensure thorough mixing with the warm saline.
  4. Transfer the serum from the refrigerated clotted tube to a large tube and add an equal volume of the warm-washed red cells.
  5. Mix and place in the refrigerator for 1-2 hours more, mixing every 30 minutes.
  6. Centrifuge the tube and check for the presence of cold antibody by adding 2 drops of the adsorbed serum to a drop of a 3% cell suspension of the patient's warm-washed cells.
  7. If agglutination persists, repeat the cold incubation of the serum with another aliquot of warm-washed red cells.
  8. If agglutination continues to be present after a second autoadsorption, pre-treat the red cells with papain. Follow manufacturer's directions.


AABB Technical Manual, 7th Edition, 1977, Page 180.

AABB Technical Manual, 13th Edition, 1999, Pages 692-693

Harmening, Modern Blood Banking and Transfusion Practices, 2nd Ed.(1989), Page 92



Authored by Peggy Schroeder Revised by Peggy Jensen


Clinical Microbiology Syllabus