(3) Proportions of serum to cells. The same general principles apply here
as discussed in paragraph 1-9. Increasing the proportion of antibody to antigen will
increase the degree of antibody coating. Two drops of serum to one drop of two
percent to five percent RBCs, a proportion of 100:1 to 40:1 in terms of packed RBCs is
commonly used. Mollison has shown that by increasing this proportion (for example, to
1000:1), sometimes weak antibodies can be detected that are not detectable in our
routine incubation mixtures. In special investigations, such as hemolytic transfusion
reaction with no antibody detectable by routine procedures, it might be useful to try
increasing the proportion of serum to cells.
(4) Incubation time. A period of 15 to 30 minutes of incubation (especially in
the presence of albumin) at 37C permits detection of most clinically significant
antibodies. Extension of the incubation period to 30 to 60 minutes may detect a few
weaker examples of antibodies that are undetected after 15 minutes of incubation.
b. Washing Phase.
(1) Washing must be rapid and uninterrupted to minimize loss of cell-bound
antibody by elution.
(2) Decant the saline as completely as possible between each washing.
Shake to loosen and resuspend the cells completely. Add the saline in a forceful
stream. An automatic washer achieves these objectives more efficiently than washing
(3) Do not cover the mouth of the test tube with the finger, or the palm of the
hand, when mixing. Serum remaining on the fingers after handling the specimen can
inactivate the antiglobulin reagent.
As little as one drop of a 1:4,000 dilution of human serum can neutralize one
drop of antiglobulin serum.
(4) Use adequate volumes of saline. When 10- X 75-mm or 12- X 75-mm
test tubes are filled at least three-quarters full of saline, three or four washings are
(5) After the final wash, discard the saline as completely as possible.
Resuspend the cells and add the appropriate amount of antiglobulin serum. Mix well,
and centrifuge. It is important that the antiglobulin serum be added immediately,
following completion of washing.