STEP 6. Add 0.1 ml of saline suspension of appropriate RBCs to each
STEP 7. Incubate in appropriate manner, according to the antibody
(9) STEP 9. Gently dislodge RBC button, and observe macroscopically for
agglutination, starting with the 1:512 tube. Record the results. Express this end point
as saline titer.
(10) STEP 10. All unagglutinated or weakly agglutinated specimens are then
washed 3 or 4 times for the AGT, if this is desired. Express this end point as
d. Technique for Master Dilution. When a diluted serum is to be studied for
reactivity against several RBC specimens, the dilutions are kept constant by preparing a
master dilution. Use larger volumes of serum and diluent, and larger tubes (if needed).
The diluted serum for each tube in each titration series can now be transferred from the
corresponding master tube. Even if only one cell is to be used, a large-volume dilution
may be easier than the 0.1-mi technique.
(1) If two or more antibodies are to be compared, it may be desirable to
evaluate more than titer. By grading the strength of reactions at each dilution, one
achieves a better understanding of the total activity, which includes strength of reaction
as well as concentration. It is essential to use the same cell suspension for all the
specimens involved, and to employ rigidly standardized technique. The numerical score
assigned to specific degrees of agglutination varies among laboratories, but it should be
uniform for all workers in any one laboratory.
(Complete or partial
hemolysis cannot easily be
quantitated. But usually
denotes a highly positive
(2) The following example of titration and scoring shows how results may be
misleading, when evaluating the relative potency of serums. The same cell suspension
is used for both serums.