b. Factors Affecting Titration.
(1) Ideally, when several serums are compared, cells from the same donor,
freshly drawn and prepared, should be used for each titration. If commercially prepared
reagent cells are used, the same genotype should be employed consistently.
(2) If the antibody is diluted with saline, the RBCs should be suspended in
saline. If a high-protein medium is used for dilution, the RBCs should be suspended in
albumin or serum.
(3) Meticulous pipetting technique is necessary for meaningful titration
results. Mouth pipetting is prohibited. Semiautomatic pipettes are recommended. A
clean pipette tip should be used for each dilution.
(4) Results should be read macroscopically. The prozone phenomenon
may produce weaker reactions in the first one or two tubes, than in the higher dilutions,
so the entire series of tubes should be evaluated, starting with the most dilute, and
ending with the most concentrated sample.
(5) Optimum incubation time, temperature, and centrifugation condition
should be determined in preliminary evaluation of the antibody. Once determined,
these should be used consistently.
(6) If serums are to be compared, the titrations should be done at the same
time. With prenatal specimens, successive samples should be stored, frozen, for
comparison with subsequent specimens. Each specimen should be tested along with
the immediate preceding sample.
Only a titer change of two tubes or more is significant.
c. Technique for Single Titration.
(1) STEP 1. Label a row of tubes according to the serum dilution, usually
1:1 through 1:512.
STEP 2. Deliver 0.1 ml of saline into the bottom of all tubes, except the
STEP 3. Add 0.1 ml serum to tubes 1 and 2 (dilutions 1:1 and 1:2).
(4) STEP 4. With a clean pipette, mix the contents of tube 2 several times;
then transfer 0.1 ml to tube 4 (1:4 dilution).
(5) STEP 5. Continue same technique through all dilutions. Remove 0.1 ml
from 1:512 tube, and save for use in further dilutions if needed.