I agree with JPCroke. People really mis-interpret the so-called rule of three. It does not mean that you must cross of every antigen three times. We see that concept a lot in recently-taught techs. A good exercise to demonstrate this is to perform a "rule-out" on your negative antibody screens. The object of crossing off is two-fold: to identify your antibody, and to rule out the coincidence of additional antibodies underlying the pattern of the first antibody. The first object is to identify the antibody. Crossing off only homozygous antigens will help if the antibody is showing dosage. Using out-of-date panels can be helpful for multiple antibody resolution. Once you have identified an antibody, you are only looking for underlying antibodies. So absolutely, you must use negative-reacting panel & screening cells to do that. At our facility, we recommend that each antigen except low frequencies be crossed off with one homozygous cell whenever possible. If it is not possible, then heterozygous cells may be used, recognizing the risk of missing an antibody showing dosage. If an antibody shows dosage, testing three heterozygous cells does not really help detect dosing antibodies. Using expired panels to identify can be helpful, but entails a risk when ruling out other antibodies: you may not see an antibody for which the antigen expression has been weakened by age; panels expire for a reason. Your AHG crossmatch will detect an incompatibility due to additional antibodies not detected.
