(4) One drop of 37C prewarmed 2 to 5 percent RBCs is added to each
tube. To row 1, add group O I adult cells; to row 2, group O i cord cells, and to the third
row, the patient's own cells. If adult i cells are available, a fourth row of dilutions is
(5) The tubes are incubated for 30 to 60 minutes and centrifuged at 37C.
The tubes are kept at 37C while reading one tube at a time rapidly, macroscopically.
Because most laboratories find it difficult to centrifuge accurately at 37C, a settling
technique may be necessary. The tubes are incubated for at least two hours and one
tube read at a time as before, but without centrifugation.
(6) After reading and resuspension of the cells, the tubes are then moved to
the next temperature (for example, 30C), and the procedure repeated. At 30C, a
settling technique (see paragraph above) will almost certainly have to be used.
(7) The tubes are finally incubated at 4C. Following 30 minutes to 1 hour at
4C, they are centrifuged at 4C. If a refrigerated centrifuge is not available, the cells
can be left to settle for at least 2 hours (even overnight) and the tubes read
macroscopically without centrifugation.
It is very important that the tests be maintained at the particular temperature
at all stages (for example, when removing tubes from the 4C refrigerator or
refrigerated centrifuge), place the rack of tubes in a container of melting ice
while reading individual tubes as rapidly as possible. If this is not done, the
cold agglutination may disperse, leading to low false values.
b. Cold autoagglutinins may be present up to a titer of 64, at 4C, in healthy
c. Cold autoabsorptions will have to be performed to detect any cold
alloantibodies (Lesson 3, Section III).
ABO AND Rh GROUPING
(1) There should be no problems in ABO and Rh typing patients with AIHA if
certain rules are followed; however, if they are not followed, serious errors can occur.
(2) The blood samples should have been collected and separated initially at
37C. The cells should be washed at 37C with warm saline. The cells are tested in the
usual fashion with anti-A, anti-B, and anti-A, B, but a negative control of 5 to 10 percent
albumin is also set up. This control should be compared with the tests, and if positive,
indicates either non-dispersed autoagglutination or spontaneous agglutination of heavily
sensitized cells in albumin.