I cannot emphasize enough proper technique in doing the washing during the elution process. We are usually concerned about too little washing and thus possibly residual reactions in the last wash. However, aggressive overwashing may remove the bound antibody resulting in a negative result.
Here is an example of anti-PP1Pk (alias anti-Tja). The mother’s panel shows an antibody to a high prevalence/incidence antigen with negative autocontrol and no lability at enzyme phase:
The neonate’s DAT was weak positive at polyspecific and IgG monospecific phases. An eluate was performed. Here is the result after washing four (4) times:
Since 2 cells in the last wash were very weakly positive, the washing was continued for a total of 9 times with the following results:
Even then there was very weak positivity in one cell, but the eluate was negative. We had washed away the attached antibodies.