d. Serum frozen for use at a later date is best distributed into several aIiquots to
avoid repeated thawing and freezing. This can denature the protein. When using
frozen serum, complete thawing and thorough mixing (including the volume in the
dropper) are required. When serum thaws undisturbed, the protein is at the bottom and
water is at the top.
a. Autologous. Each procedure used for antibody detection or identification
should include a tube containing the serum and washed cells of the person being
tested. These autologous controls can then be compared to the tubes containing the
The patient's serum is tested against his own red cells just as if they were
b. Reagent Red Blood Cells. Because the serum of all patients and donors
must be tested for the presence of unexpected antibodies, reagent RBCs should be
observed for possible deterioration. When serum or cells under test are subjected to
physical or chemical alteration (as in enzyme techniques or
two-mercaptoethanol treatment), reagents of known behavior must be included as
c. Control Red Blood Cells. When unknown cells are tested with known
antiserums, both positive and negative control cells should be included. The positive
control should have weak expression of the antigen to be certain the serum detects
weak as well as strong antigens. For example, the positive control for anti-Jka serum
should be heterozygous Jk (a+b+) rather than homozygous Jk (a+b-) to be certain the
antibody detects a single dose of the antigen because the antigen properties of the cells
being tested are unknown.
1-31. ANTIBODY DETECTION
a. A routine procedure for antibody detection should be developed and be
available in writing. Each member of the staff should know, and follow the directions as
written. Example: Antibody detection.
(1) STEP 1. Label tubes appropriately for the screening cells and an
autocontrol, (number of tubes will vary depending on type of screening cell used).
STEP 2. Use the techniques described in paragraph 1-25.
(a) Saline technique. (Incubated at room temperature (RT) for 30
Albumin technique incubated at 37C for 30 minutes.