ABSTRACT: Patients with recurrent spontaneous abortions have been sucessfully treated in many centers with third-party immunization directed to a putative TLX antigen system. This immunotherapy requires the screening of a large number of donors to match the patients' red blood cell (RBC) phenotype and has the potential risks associated with transfusions from 30 to 50 donors. Our modified approach to third-party immunization is to use irradiated frozen-stored purified lymphocytes pooled from five normal donors. Mononuclear cells from normal donors are obtained in a cell separator. After sedimentation and Ficoll-Hypaque separation, the cells are stored in liquid N2. The RBC depletion of the final preparation is of the order of 5 to 6 logs, theoretically decreasing the need for RBC phenotyping except for the Rh system. Using a highly sensitive fluorescence-activated cell sorter technique and an ADCC assay, we found that ABH, Rh, Fya Fyb, JkaJkb, MNS, and Kell antigens are either not expressed by cryopreserved human mononuclear cells, or, if so, they are below the level of detection of these highly sensitive assays. We conclude that the use of pooled frozen mononuclear cells is an adequate alternative for immunotherapy. It decreases the transfusion risks associated with exposure to a large number of donors and the need for RBC phenotyping, making this modality of treatment more accessible.